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Question: <p>I was wondering why a denatured protein isn't able to fold back into it's native form again. <br>
Because a polypeptide before it's folded has a enthalphy and entropy drive to do so. What does the denaturation process do whith this favouring force to fold into the native structure. <br>
In my opinion I can't see the differences between the unfolded protein (direct after translation) and the denaturated protein. </p>
Answer: <p>In short: the unfolded state is a high-energy state of the protein, which will move towards lower-energy states. Some of these states are the folded protein, while others states are denatured protein (forming "wrong" interactions with itself or other proteins). Energy barriers between these states keep the protein in the denatured state even though the folded state might me more favorable. </p>
<p>Or in other words: it's trapped in the denatured state because of kinetics, even though thermodynamics would favor the folded state. </p>
<p>A nice figure to illustrate this: <a href="http://www.ghrnet.org/index.php/jbmbr/article/viewFile/1027/1397/6798" rel="nofollow">http://www.ghrnet.org/index.php/jbmbr/article/viewFile/1027/1397/6798</a></p>
<p>If you go into a little bit more detail, you will find that a lot of proteins (especially larger ones) are usually folded by chaperones. These helper proteins will bind to the emerging protein chain to prevent it from aggregating. In a sense they guide the protein to a folded state. In a similar way, chaperones can help proteins overcome the energy bariers that normally prevent it from going from an aggregated state to a folded state. </p>
<p>This review in Science is very complete, but also contains some nice figures that give you an idea of what happens:
<a href="http://science.sciencemag.org/content/353/6294/aac4354.long" rel="nofollow">http://science.sciencemag.org/content/353/6294/aac4354.long</a> / </p>
<p>If you're a pirate: <a href="http://science.sciencemag.org.sci-hub.bz/content/353/6294/aac4354.long" rel="nofollow">http://science.sciencemag.org.sci-hub.bz/content/353/6294/aac4354.long</a></p>
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https://biology.stackexchange.com/questions/51408/why-denatured-proteins-cant-fold-back-in-their-native-form
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Question: <p>Suppose I have number of PDB files of proteins. How can I get the number of folds present in these proteins? Is the fold count derivable from the PDB files? If so, how?</p>
Answer: <p>There are various ways that you could do this.</p>
<ol>
<li><a href="http://www.cathdb.info/" rel="nofollow">CATH</a> is a hierarchical classification system</li>
<li><a href="http://scop.mrc-lmb.cam.ac.uk/scop/" rel="nofollow">SCOP</a> is another such system with a different hierarchy</li>
<li><a href="http://ptgl.uni-frankfurt.de/" rel="nofollow">PTGL</a> is the protein topology graph library</li>
<li><a href="http://bioinf.mii.lu.lv/tops/" rel="nofollow">Tops motif</a> will scan PDB files and match patterns against it</li>
</ol>
<p>How you actually apply these tools or lookup within these systems is documented on their websites. However, it depends on what you want to do - look up published structures, or new experimental data.</p>
<p>I should point out that writing software from scratch to determine the fold of a protein may be tricky.</p>
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https://biology.stackexchange.com/questions/51768/how-to-calculate-the-number-of-folds-present-in-a-protein
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Question: <p>While peptide bonds usually adopt the <em>trans</em> conformation, peptide bonds to proline can exist in either <em>cis</em> or <em>trans</em> conformation. The isomerization between <em>cis</em> and <em>trans</em> is slow, and has been shown to be the rate-limiting step in folding of certain proteins. </p>
<p>What methods can be used to determine the conformation of a specific proline in a protein? </p>
Answer: <blockquote>
<p>Sarkar et al.(2007) Proline cis-trans Isomerization Controls Autoinhibition of a Signaling Protein. <em>Molecular Cell</em> <strong>25</strong>, 413–426 (DOI 10.1016/j.molcel.2007.01.004), available <a href="http://chem.rutgers.edu/~babis/Publications/Sarkar_Molcell.pdf">here</a>. </p>
</blockquote>
<p>The authors report on the interactions between two SH3 domains of the Crk adaptor protein. Basically they find that the linker which tethers these two domains contains a proline which interconverts between the <em>cis</em> and <em>trans</em> conformations, and that this interconversion determines the interaction between the two domains. They follow isomerisation by using <a href="http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopy_of_proteins">NMR</a>.</p>
<p><em>Cis-trans</em> peptidyl-prolyl isomerization is a subtle change, and I don't think that you will find a way of following it in a specific protein that is simpler than this, unfortunately.</p>
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https://biology.stackexchange.com/questions/5178/determining-if-a-specific-proline-is-cis-or-trans-in-the-protein
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Question: <p>Initially, I was going to ask how many proteins were possible. But, while researching the question, I learned the word proteoform and have been reading a lot of stuff I don't really understand, but from I do think I get, the word "proteoform" was created because sometimes the "typical" or "canonical" forms of a "protein" can take different forms, or "proteoforms" based on environmental and possibly other factors, despite being coded for with the same DNA. So, a "protein" will often (always?) have several different "proteoforms". As I understand it, it is part of the protein-folding problem, and the complexity is huge. I was trying to see if anyone has calculated or figured out what would be needed to calculate how many proteoforms were possible, but I don't find anything. I imagine the number is tremendous, I just wanted to know how tremendous. So, how many proteoforms are possible? If that's not known, what would be needed to know this? Thanks.</p>
Answer:
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https://biology.stackexchange.com/questions/110615/how-many-proteoforms-are-theoretically-possible
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Question: <p>Do they fold independently of each other or synergistically? If we express these globular domains separately, would their structures remain the same?</p>
<p>I came up with an easy way to test it. Many RNA viruses like HIV and coronaviruses encode multiple proteins in one peptide which folds into a polyprotein with multiple globular domains. The globular domains are then separated by the viral protease. Can we express these proteins separately and compare their structures with native ones?
<a href="https://i.sstatic.net/825nh.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/825nh.jpg" alt="enter image description here" /></a>
<a href="https://i.sstatic.net/9wvDt.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/9wvDt.jpg" alt="enter image description here" /></a>
This question is not a mere curiosity. Many proteins have multiple globular domains which makes structure determination difficult because these domains are very loosely joined together. It would be much easier if we can solve the globular domains individually instead of solving the whole protein at once.</p>
Answer: <p><strong>A broad interpretation of the question</strong><br>
The question refers to the “folding” of proteins. This term — and the associated biological problem — is often used in the rather specific sense of the attainment of a structure with a particular thermodynamic minimum free energy. I take the intent of the OP (original poster) to be whether, or to what extent, one component of a polyprotein influences the structure adopted by another by physical association. I shall extend the question to that as I think an attempt to answer it will be more interesting and rewarding.</p>
<p><strong>The general and the specific</strong><br>
I do not think one can assume that there will be a single answer to the question that will be true for all polyproteins. Nature is seldom so obliging, and there is no guarantee that what may be true for a polyprotein translated from a viral RNA will also be true for one translated from a cellular RNA. Even within these categories it would be prudent to consider things on a case by case basis.</p>
<p><strong>The answer in summary</strong><br>
Comparisons of the sort suggested have been made in certain cases or are implied by the accumulated work in others. In the polyproteins of both RNA virus and cellular biosynthetic complexes the evidence suggest independent folding in many cases. However in some viral polyproteins, particular pairs of protein domains may be have as a single folded protein in precursors to proteolytically cleaved subsequent forms.</p>
<p><strong>Viral polyproteins</strong><br>
Comparisons of the sort suggested by the OP have, in fact, been performed, although not on complete viral polyproteins — rather restricted to examining possible interactions of particular subsections of the latter. I can see two reasons for this:<br>
<em>Practical Difficulty.</em> I am not aware of any viral polyprotein that has had its total structure determined. This may be because large protein assemblies are generally difficult to crystalize, or because the flexibility of the linkers may mean most do not adopt a single structure, or because even if they do they may not maintain it for any length of time before proteolysis occurs.<br>
<em>Insufficient interest in the question.</em> I suggest that most virologists would assume that the sole or primary reason for synthesis of RNA virus proteins as a polyprotein is that the mechanism of eukaryotic translational initiation precludes polycistronic mRNAs (in all but a few cases not relevant here), and the way the linkers space the protein domains suggests that they fold independently of one another. Furthermore, the unrelated nature of many adjacent protein domains (polymerases, proteases, structural proteins) — and the non-conserved organization of neighbours — gives no reason to suppose that they interact in any way. There is a long history of the crystallization of individual RNA viral proteins expressed from artificial constructs including only the open reading frame of the viral RNA, as exemplified most recently by the expression of Covid proteins for research and vaccine development. This also suggests that individual proteins fold independently.</p>
<p><strong>Comparison studies on partial viral polyproteins</strong><br>
Nevertheless, it has been recognized that — and there has been considerable interest in the fact — the viral polyprotein system has been refined and adapted to the infection. In some cases, for example, there is a process of protein maturation by successive proteolysis, with intermediates having functionally different properties to the mature end-product. For this and other reasons structural analysis and comparisons of parts of the polyprotein have been performed with different viruses and proteins, some of which were reviewed by [Yost and Marcotrigiano in 2013].(<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660988/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660988/</a>)<br>
<a href="https://i.sstatic.net/XCabY.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/XCabY.png" alt="Poliovirus 3CD precursor" /></a><br>
One example (from this review) is the 3C/3D precursor of poliovirus, in which the two components are well separated (above) and, to quote, “Overall, the structures of 3C and 3D alone are very similar to their counterparts in the 3CD precursor.“</p>
<p>The preceding result may be the one anticipated, but the nsP2/nsP3 precursor of Sindbis virus illustrates a quite different situation. Here the two proteins components interact strongly in the precursor (below), suggesting that after proteolysis a rearrangement of the structures or interaction with some other protein might occur. Suffice it to say that the situation is complex and remains to be resolved. However a comparison of with the structures predicted by alphaFold might be informative.<br>
<a href="https://i.sstatic.net/Cr9NE.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Cr9NE.png" alt="Sindbis virus nsP23 precursor" /></a></p>
<p><strong>Cellular Polyproteins</strong><br>
The situation with cellular polyproteins is rather different. These relate synthetic pathways in which the enzymes catalysing successive steps are organized into a pipeline. In the (relatively few) examples the eukaryotic version is a polyprotein, whereas the prokaryotic equivalent is a multi-enzyme complex of similar, but distinct, components. In contrast to the viral polyproteins, there seems no a priori reason for a polyprotein in this case, and, indeed, this is not subject to (natural) proteolytic cleavage.</p>
<p>Polyproteins of this type have been crystallized only relatively recently, although there are several structures of individual components and prokaryotic equivalents. I shall consider two examples.</p>
<p>The first is enzymes of the <a href="https://en.wikipedia.org/wiki/Shikimate_pathway" rel="nofollow noreferrer"><strong>shikimate pathway</strong></a>, responsible for the synthesis of aromatic amino acids. In bacteria, such as <em>Escherichia coli</em>, this comprises seven individual enzymes, but in lower eukaryotes such as <em>Neurospora crassa</em> a polyprotein of five of these, <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1164546/" rel="nofollow noreferrer">the <em>arom</em> complex</a>, is employed. (Higher animals lack the ability to make aromatic amino acids, and plants use separate enzymes encoded by the chloroplast genome.) The structure of the arom complex polyprotein was determined for the thermophilic fungus, <em>Chaetomium thermophilum</em>, by <a href="https://www.nature.com/articles/s41589-020-0587-9" rel="nofollow noreferrer">Verasztó <em>et al</em>.</a> A feature of this polyprotein is that the individual enzyme domains move and function independently of one another with little effect from their linkages. Although many structures have been reported for the individual enzymes of the complex, these differ between species, and I can find none for <em>C. thermophilum</em> in the protein data bank. Hence the exact test proposed by the OP has not been carried out, but the indirect evidence suggests independent folding. <a href="https://doi.org/10.26508/lsa.202101358" rel="nofollow noreferrer">A structure</a>, from <em>Candida albicans</em>, has subsequently been reported.</p>
<p>The second example of a cellular polyprotein is the enzymes of the <strong>fatty acid synthase</strong> pathway in mammals, and the homologous polyketide synthases in bacteria (only few of which synthesize fatty acids). The first such structure was reported by <a href="https://www.science.org/doi/10.1126/science.1163785" rel="nofollow noreferrer">Maier <em>et al.</em> in 2008</a>. The porcine complex is shown below, and has a more open and flexible design than the <em>arom</em> complex, that seems to have allowed insertion and deletion of different catalytic domains during evolution. Again, this argues in favour of independent folding of individual components, although I am not aware of direct comparison with individually expressed fragments. I suspect that there might be structures available to do this, although I haven’t had the time to examine this further myself.</p>
<p><a href="https://i.sstatic.net/v6mme.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/v6mme.png" alt="FAS complex" /></a></p>
<p>I would conclude by noting that although the individual components of these cellular polyproteins interact with one another to cooperate in the biosynthesis, the flexibility they exhibit allows transfer of the growing substrate from one component to another.</p>
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https://biology.stackexchange.com/questions/114078/if-a-protein-has-multiple-globular-domains-with-flexible-peptide-linkers-in-betw
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Question: <p>I have a list of pairs of gene symbols who interact (putatively) and would like to assign each pair a score (and record other details) based on the predicted Protein-Protein Interaction (PPI). The existing PPI webservers I've looked at (<a href="http://cluspro.bu.edu/home.php" rel="nofollow">ClusPro</a> and <a href="http://www.bonvinlab.org/software/haddock2.2/haddock.html" rel="nofollow">HADDOCK</a>) require that I use <a href="http://www.rcsb.org/pdb/home/home.do" rel="nofollow">PDB</a> ID's as input. Unfortunately, PDB ID's don't always exist for the gene symbols I'd like to compare. I believe I need either:</p>
<ol>
<li>a way to generate PDB files myself (perhaps with a folding program?) and then connect/upload them to a PPI webserver.</li>
</ol>
<p>or</p>
<ol start="2">
<li>a way to find the most similar sequence that does have a PDB ID and upload it to a PPI webserver. </li>
</ol>
<p>Am I understanding my problem correctly? Are there resources that can help with 1 or 2?</p>
Answer: <p>This may or may not be possible, depending on what proteins you are considering. Generating a PDB file means predicting the structure of the protein. There are no methods for predicting protein folding accurately from plain sequence data, so you will need <em>some</em> experimental data on the structure of your proteins.</p>
<p>If your proteins have not been structure determined, the next best thing is some kind of <a href="https://en.wikipedia.org/wiki/Homology_modeling" rel="nofollow">homology model</a>, that is, a prediction of your protein structure based on a known structure of a homologous protein. These can be more or less accurate, depending on how close the homolog protein is. If specific domains are interacting, it may be enough to create a model of the domain only.</p>
<p>There are online tools available for homology modeling, for example <a href="http://swissmodel.expasy.org/" rel="nofollow">swiss-model</a> from the expasy site. This is not an automated process though --- creating an accurate model requires some skills. You should read the publications describing these tools and make sure you understand the process and error souces, and perhaps contact an expert if in doubt. Good luck!</p>
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https://biology.stackexchange.com/questions/42609/how-to-predict-protein-protein-interactions-from-a-pair-of-gene-symbols
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Question: <p>For <strong>DNA</strong> one can distinguish between</p>
<ul>
<li><p>protein-coding DNA sequences, i.e. nucleic acid sequences inside DNA (vs. <a href="https://en.wikipedia.org/wiki/Noncoding_DNA" rel="nofollow noreferrer">non-coding sequences</a>)</p></li>
<li><p>DNA sequences that do not code for proteins but are transcribed into functional RNA</p></li>
<li><p>non-transcribed DNA sequences that have other (e.g. regulatory or structural) functions</p></li>
<li><p>non-transcribed and non-functional DNA sequences (‘junk DNA’)</p></li>
</ul>
<p>The relative proportions of these seem to <a href="https://en.wikipedia.org/wiki/Noncoding_DNA#Fraction_of_noncoding_genomic_DNA" rel="nofollow noreferrer">depend heavily on the organism</a> and are only roughly known.</p>
<blockquote>
<p>What I wonder is whether there are protein sequences (i.e. amino acid sequences inside a protein) that have no functional purpose.</p>
</blockquote>
<p>A non-functional protein sequence might be a sequence that is neither effective to the outside (e.g. as a site for <a href="https://en.wikipedia.org/wiki/Post-translational_modification" rel="nofollow noreferrer">post-translational modification</a>) nor to the inside (e.g. in folding). But even those parts of the protein might contribute structurally by their sheer presence and mass which is important when considering proteins not only as chemical but also as somehow mechanical devices. This might be called its structural purpose.</p>
<p>Assuming that each part of a protein has either a functional or only a structural purpose the question arises what the relative proportions of functional vs. only structural parts are.</p>
Answer: <p>This question is unanswerable as, if a protein exists as a physical entity in a cell it is possible to demonstrate it has a functional or structural role, but it is logically impossible to demonstrate it has no such role. The best one can say is that <em>“it has no known function (role)”</em>.</p>
<p>That said, in the course of evolution following gene duplication and divergence it is theoretically possible that <em>on the way to acquiring a new functional role</em> there is a stage at which a protein has no such role. One might be able to identify such proteins in a gene family like the <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543078/" rel="noreferrer">globins</a> by examining a range of species, as has been done for the globin genes. I am not aware of any such studies.</p>
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https://biology.stackexchange.com/questions/68948/do-non-functional-junk-protein-sequences-exist
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Question: <p>I am not very familiar with the experimental procedure of x-ray crystallography except that it involves the very delicate matter of producing crystal that contain proteins and then diffracting rays through it to get a pattern that tells us about the shape of the protein.</p>
<p>I am curious though when you crystallize a protein that usually stays in the cellular fluid, does it go through any conformational or size changes. For instance isn't there some pressure applied by the water that would potentially result in protein being smaller then what it might be under less pressure. Or what about hydrophobic\philic effects that play an important role in protein folding. Of course once the protein is folded it is bonded through interactions stronger than hydrophobicity so it is not that delicate. But still a complete change of surrounding environment should count as a big change, should it not? So are there any theoretical or experimental explanations as to whether the protein changes size and\or shape during crystallization? References to both experimental and theoretical work are very welcome. Although I guess experimental evidence would make more sense in this matter since usually potentials are optimized to account for the crystal structure to be the minimum of the energy so it I can't see how theoretical works could potentially help to understand this issue. I guess one way would be to take a native protein structure determined by X-ray and run it through AB initio molecular mechanics where potentials do not depend on parameters obtained from the native states of proteins on PDB database. I don`t know how theoretically sound that would be though. </p>
Answer: <p>Protein crystals are not like crystals of more commonly found substances like salt [NaCl] or diamond [carbon only.] These materials do not include other atoms in their crystal structures. For instance, a crystal of NaCl will contain sodium ions and chloride ions. X-ray crystallography of that material will, after mathematical processing, show electron density peaks of two varieties, each easily distinguished by intensity as either a sodium ion or chloride ion. Any other electron density peaks will be few and far between, as well as clearly identifiable as an interloper.</p>
<p>Because most proteins have hydrophilic regions on the exterior surface of the structure, crystals of proteins actually contain a considerable fraction of water molecules within the crystal itself. These water molecules are part of the crystal because they are interacting with those hydrophilic residues on the tertiary surface of the protein, both by hydrogen bonding, in some cases, and less specific polar interactions in other cases. This is at least one of the reasons why obtaining a crystal of any random protein is not at all a routine endeavor. The large proportion of water in these crystals make them very fragile once they do form, as well as not necessarily likely to form in the first place.</p>
<p>If you go into the technical literature to look at the electron density maps that the more commonly diagrammed structure maps are derived from, you'll be able to actually trace water molecules surrounding the individual protein molecules.</p>
<p>In fact, in the days of development of protein crystallography, assigning correct specific atoms to the various electron density peaks was a decidedly non-trivial task.</p>
<p>Effectively, despite being a crystal, the microenvironment experienced by the protein within the crystal is very much like that in an aqueous environment. The hydrophobic interactions & hydrophilic interactions will not be very different from those in solution.</p>
<p>That is, the crystalline state achieved is not, in fact, "a complete change of surrounding environment" to use the phrase that you've used in your post.</p>
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https://biology.stackexchange.com/questions/46205/proteins-in-water-vs-proteins-in-crystal
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Question: <p>In my lab we are trying to extract spatial features from protein structures. The software we develop makes use of CUDA for all heavy number-lifting, thus we are limited by the GPU's memory (12GB). Using standard voxel-based 3D-representations proved to be too memory hungry, hence we are trying to find a way to reduce the dimensionality to 2D, while preserving as much spatial information as possible. Our absolute goal is to preserve spatial colocalisation of different chains and secondary structures, to understand how important that is during folding. Standard techniques, such as PCA and PCoA on euclidian distances between amino acids, seem to preserve too little information on chain folds. We believe there should be a better way of doing that, but, having little experience in structural biology, we struggle to find any relevant work regarding this issue. Can you recommend any methods or relevant readings? </p>
Answer:
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https://biology.stackexchange.com/questions/52583/reduce-protein-structure-representation-dimensionality
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Question: <p>By default, <code>pymol</code> seems to grab the number of cores on the system for rendering. How can I force it to only use one core?</p>
<p><strong>Motivation:</strong></p>
<p>I have a large collection time-series of of coordinate data from a computational protein folding experiment. I'd like batch render the generated pdb files into a movie in a programmatic way. Pymol however likes to eat up as many cores as it finds available. I'd like to retain some control of the computational burden (required for some computational cluster jobs). How can I force pymol to only use one (or n) core(s)?</p>
<p><strong>Solution:</strong></p>
<p>While the question was closed (as it was decided to not fit into biological criteria of the site) future visitors might find it useful to know the answer:</p>
<pre><code>set max_threads, 1
</code></pre>
<p>The solution was found in a <a href="http://biostars.org/post/show/44065/turn-off-multithreading-in-pymol" rel="nofollow">Biostars question</a>.</p>
Answer:
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https://biology.stackexchange.com/questions/2088/turn-off-multithreading-in-pymol
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Question: <p>I was reading this article: "<a href="https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&uact=8&ved=0CDgQFjAE&url=http%3A%2F%2Fwww.researchgate.net%2Fpublication%2F12196921_Overview_of_vector_design_for_mammalian_gene_expression%2Flinks%2F0912f50b859ea95140000000&ei=ygFcVKzCHteUsQSpm4LoAw&usg=AFQjCNHJpnMNYd2BQzxgF8-08QC2Mw9dTQ&sig2=3FJpi89PA0wj3WKu3eJmfQ" rel="nofollow">Overview of vector design for mammalian gene expression.</a>" for an explanation of why mammalian cell lines are used for expressing cloned genes, and one of the reasons given is that "DNA cloned from higher eukaryotic cells is readily expressed since the signals for transcription, mRNA processing, and translation are conserved in higher eukaryotic systems." What does it mean for the signals to be "conserved," and why does this make mammalian cells a better host than other types of cells?</p>
<p>Follow-up question, in case the answer to that question doesn't make it clear: the article also says the machinery for protein folding and assembly are "conserved" - what does that mean (if not the same thing as in the first question) - and again, why does this make mammalian cells a superior choice of host for expressing cloned genes?</p>
Answer: <p>I concur with @Bez but wish to elaborate on the meaning of 'conserved'. It is generally used in the context of evolution. A conserved characteristic or gene or protein means that it has 'survived' a long time without being altered. As @Bez mentions, certain parts of the protein machinery in eukaryotes is very different from prokaryotes, the latter being considered to have arisen much earlier in evolution. For example, some post-transcriptional processes (e.g. splicing <a href="http://www.majordifferences.com/2013/10/difference-prokaryotic-and-vs.html#.VFyoRk0cTs0" rel="nofollow">and many more differences</a>) only occur in eukaryotes. But there are many similarities as well, such as the basic use of mRNA etc (see <a href="http://www.ncbi.nlm.nih.gov/books/NBK22531/" rel="nofollow">book section</a>). The latter are examples of conserved mechanisms. In the context of your question, it means that when you take eukaryotic DNA to express eukaryotic proteins it doesn't really matter which cell or host organism you take as it will be transcribed and folded correctly. For example, yeast is often used to express DNA from higher organisms as its protein synthesis is much the same across various eukaryotic species. However, expressing eukaryotic DNA in prokaryotes likely ends up in misfolded proteins without proper post-transcriptional modification and should be avoided. </p>
<p>As to your follow-up question: Folding of proteins happens in the endoplasmatic reticulum (ER) in eukaryotes (<a href="http://en.wikipedia.org/wiki/Endoplasmic_reticulum" rel="nofollow">wiki</a>), a cell organelle absent in prokaryotes as they lack a nucleus and associated organelles. Again, expression of eukaryotic genes in a prokaryotic expression system will end up in mis-folded proteins. </p>
<p>To get back to your overall question - the 'signals': DNA transcription, splicing, protein synthesis, post translational modification and folding are all regulated by signals, such as specific DNA codons to start and stop transcription, RNA codes for splicing and start translation and protein tags that are placed on specific amino acid combinations. These signals are well conserved across eukaryotic species. </p>
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https://biology.stackexchange.com/questions/23783/what-does-it-mean-for-the-signals-for-transcription-and-translation-to-be-conse
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Question: <p>The field seems extremely divided on the debate. On one hand, artificial experiments have suggested that synonymous mutations don't correlate with gene expression but rather, the mRNA 5' structure is the most important <a href="http://www.ncbi.nlm.nih.gov/pubmed/19359587">1</a>. On the other hand, genome wide analysis suggests that tRNA biases are better associated with high expression <a href="http://www.ncbi.nlm.nih.gov/pubmed/20403328">2</a>. What other works balance out this discussion?</p>
<ol>
<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/19359587">Coding-sequence determinants of gene expression in Escherichia coli</a></li>
<li><a href="http://www.pnas.org/content/107/8/3645.short">Translation efficiency is determined by both codon bias and folding energy</a></li>
<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/20403328">An evolutionarily conserved mechanism for controlling the efficiency of protein translation</a></li>
</ol>
Answer: <p>This is an excellent question! To my knowledge, there hasn't been a definite answer yet. Recently, I did tons of research on which factors influence protein expression and you should definitely check out the following questions which I asked: </p>
<ol>
<li><p><a href="https://biology.stackexchange.com/q/1/28">What is the criticality of the ribosome binding site relative to the start codon in prokaryotic translation?</a></p></li>
<li><p><a href="https://biology.stackexchange.com/q/166/28">What determines a successful protein expression in E. coli?</a></p></li>
</ol>
<p>I answered my second question by posting a concise version of <a href="http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&ved=0CFoQFjAF&url=http://mpec.ucsf.edu/pdfs_new/Pubs_77.pdf&ei=xkMHT-XmKqbdiAKv4JSHCQ&usg=AFQjCNFcrXypViPECyKyeUvRM2JmZtidGA&sig2=0BtAug3bPEHhzZDqzuFUjA" rel="nofollow noreferrer">a paper</a> from DNA2.0 (a gene synthesis company) that discusses the protein expression. It summarizes all the important factors (RBS site, codon frequency match, 5' mRNA secondary structure), but it doesn't discuss the extend to which they influence expression. </p>
<p>There is a nature paper from Voigt lab about <a href="http://www.nature.com/nbt/journal/v27/n10/full/nbt.1568.html" rel="nofollow noreferrer">RBS calculator</a>, which takes into account only the 5' mRNA secondary structure and it doesn't discuss at all the codon-optimization. The calculator was good, but far away from perfect: there is a 50% chance of predicting an expression level within a 2-fold range of the target one.</p>
<p>On the other hand, an <a href="http://www.ncbi.nlm.nih.gov/pubmed/16756672" rel="nofollow noreferrer">older paper from DNA2.0</a> argues that the mRNA is covered with ribosomes practically all the time, so the secondary structure of the mRNA should have little effect. Besides, an actively translating ribosome can break up stem-loop structures.</p>
<p>In my opinion, we are still far off from predicting RNA secondary structure formation, although it might be highly implicated in protein expression. From what I read, I think that it is equally crucial that there is no strong secondary structure at the 5' end of the mRNA, the RBS sequence is close to the consensus one and it it appropriately spaced upstream of the start codon, and the first 60-100 codon frequencies match the frequency of your heterologous host. </p>
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https://biology.stackexchange.com/questions/1152/which-is-more-important-for-protein-expression-mrna-structure-or-codon-optimizat
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Question: <p>An acquaintance provided me with <a href="http://www.ncbi.nlm.nih.gov/pubmed/15321723" rel="nofollow">this article</a><sup>1</sup>. I can't understand for sure what it is about. </p>
<p>My acquaintance said that it proves that time for generation of even the simplest proteins is on a larger timescale than evolution history (billions of years). </p>
<p>I'm asking you, is that a valid conclusion to be made from this paper? What is in this article that might drive someone to that conclusion?</p>
<ol>
<li>Axe,D.D. (2004) Estimating the prevalence of protein sequences adopting functional enzyme folds. J. Mol. Biol., 341, 1295–315. <a href="http://www.ncbi.nlm.nih.gov/pubmed/15321723" rel="nofollow">http://www.ncbi.nlm.nih.gov/pubmed/15321723</a></li>
</ol>
Answer: <p>I think your acquaintance is trying to fit real science to some of his personal beliefs (that are obviously wrong). </p>
<p>If you read the article you'll see that it's not about evolution at all, but about protein folding and what proportion of possible sequences gives a working protein. It turns out random sequences are not that likely to fold, which leads to some conclusions about the probable mutational pathways to folded proteins. </p>
<p>The argument of your friend is sort of analogous to the infinite random monkey theorem. If you put a monkey behind a typewriter and wait long enough, eventually he'll write the complete works of Shakespeare. This doesn't mean we waited billions of years for this monkey to type it up, because the world doesn't work like that. There was just a guy with a brain. </p>
<p>In case of the proteins, evolution doesn't work by generating newly (random) 300 residue proteins that work 1 time in a billion. Probably in one generation a copy of the protein is made with the same function, and during many generations the function of this protein drifts towards a new function, while all the intermediate proteins stay folded because they're not that different. </p>
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https://biology.stackexchange.com/questions/51670/can-estimating-the-likelihood-of-protein-sequences-adopting-functional-enzyme-fo
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Question: <p>I'm wondering if there is some threshold in size or a specific structural property that determines if a small protein or large peptide would cause an immune reaction. </p>
<p>Context: there are a number of drugs being developed based on antibody mimetics (small protein scaffolds with stable folding). These tend to have a size around 50-60 aminoacids (6-7 kD). These tend to be much easier to design than human / humanized antibodies. However, it is not uncommon for these to cause anaphylaxis, especially if administered repeatedly. It may be good to somehow design a version of a small antibody mimetic which itself does not cause an immune reaction.</p>
<p>Are peptides around 10-20 aminoacids typically immunogenic? What about slightly larger ones up to 30 aminoacids? Does it matter if they have a stable secondary or tertiary structure?</p>
Answer: <p>Edited to delete off-topic section: </p>
<p>Most (all?) proteins are ultimately immunogenic. You can't really design your way around immunogenicity, which is a good thing, because <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956326/" rel="nofollow noreferrer">otherwise we'd all be dead from viruses</a>. </p>
<p>Obviously not all proteins are equally immunogenic, but <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062386/" rel="nofollow noreferrer">basically all protein therapeutics are immunogenic at some rate</a>. One thing that seems positively associated with immunogenicity in that review is quaternary structure, but it's quantitative not qualitative.</p>
<p>Some of the commonly used synthetic antigens used in biochemistry such as <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC253590/" rel="nofollow noreferrer">HA C-terminus</a> (24 residue) or <a href="https://www.sigmaaldrich.com/catalog/product/sigma/f3290?lang=en&region=US" rel="nofollow noreferrer">FLAG</a> (8 residue) are quite small. </p>
<p>More generally, <a href="https://academic.oup.com/intimm/article/12/3/375/775132" rel="nofollow noreferrer">this study finds that even 3-5 residue peptides can be sufficient</a> to raise immune responses. So <strong>likely any polypeptide is potentially immunogenic</strong>.</p>
<p>Such small peptides are unlikely to have meaningful secondary/tertiary structure. My guess is that that you would have to have a pretty structurally strange protein to avoid any potential immune response. However if you had an insoluble or somehow sequestered protein you might be able to avoid it, but I'm not an expert so I can't say how.</p>
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https://biology.stackexchange.com/questions/92562/what-determines-if-a-small-protein-large-peptide-is-immunogenic
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Question: <p>The book, <em><a href="https://books.google.co.in/books?id=xYmcAQAAQBAJ&lpg=PA6&ots=7fejD9SWmI&dq=peripheral%20dogma%20(bioinformatics)&pg=PA6#v=onepage&q=peripheral%20dogma%20(bioinformatics)&f=false" rel="nofollow noreferrer">Introduction to Bioinformatics</a>, by Arthur M. Lesk, 3rd edition; Oxford; low-price-edition</em>; in its chapter-1 (introduction), page-no. 6 ; provided a paragraph, entitled <strong>"Dogmas: Central and Peripheral"</strong>. </p>
<p>As-if; there exist a contrasting term for <a href="https://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology" rel="nofollow noreferrer">central dogma</a>. </p>
<p>But while thoroughly going through that paragraph (and other chapters linked(mentioned) in that-paragraph; I couldn't found the mention of the term "peripheral dogma" or such. </p>
<p>From the paragraph; it looks like the term "peripheral" has been used to indicate all information-flows other-than <a href="https://en.wikipedia.org/wiki/Genetic_code" rel="nofollow noreferrer">genetic code</a>s, and including Junk-DNA and protein-folding-variations. No mention of epigenetic codes found.</p>
<p>In Web too; I could found only 1 <a href="http://eclass.uoa.gr/modules/document/file.php/D464/%CE%94%CE%B9%CE%B1%CE%BB%CE%AD%CE%BE%CE%B5%CE%B9%CF%82%20I.%20%CE%95%CE%BC%CE%AF%CF%81%CE%B7/0.intro.pdf" rel="nofollow noreferrer">website</a> used the term "peripheral dogma" in context with biology. which, too, does-not mention the definition clearly. </p>
<hr>
<p>So, What is peripheral dogma?</p>
<hr>
<p>P.S. this is purely a terminology question; which is looking for authoritative reference about usage of a term.</p>
Answer: <p>I think the author was just trying to make a point that while the "central dogma" is indeed key, there is more to it than that and that understanding of molecular biology has advanced beyond being simplified to that one tenet. Without having the passage in front of me, the ideas you gleaned from the paragraph make sense, though others such as epigenetics seem like they would fit as well.</p>
<p>As you've noticed, this term is difficult to find anywhere else, it isn't a standard term and therefore doesn't really have any meaning unless it is defined in the source. I wouldn't recommend using it yourself unless you plan to define it. The Wikipedia page for <a href="https://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology#Transfers_of_information_not_explicitly_covered_in_the_theory" rel="nofollow noreferrer">central dogma</a> that you linked has a whole section on information transfer not included in the "central dogma" - I think you can safely assume the author was referring broadly to these items.</p>
<p>This all may be a bit of a joke, as well, given the definition of "dogma" and it's somewhat inappropriate use in the context of the central dogma of molecular biology - again, the Wikipedia article you linked talks about the problematic use of the word "dogma" in this scientific context.</p>
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https://biology.stackexchange.com/questions/53097/what-is-peripheral-dogma
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Question: <p>I am studying how transmembrane proteins are made and I have read that proteins that are destined for the plasma membrane are initially in the ER membrane and do not get translocated into the ER lumen unlike other proteins.</p>
<p>I was wondering when a transmembrane protein destined for the cell membrane is in the ER membrane, is in its native form (e.g. final, folded form?). I am asking this because I read that the ribosomes are on the ER membrane, and that protein translocation through the ER membrane occurs co-translationally, meaning that it's occurring as the mRNA is being translated. Would the protein get folded as it's moving through the ER membrane?</p>
<p>Any insights are appreciated.</p>
Answer:
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https://biology.stackexchange.com/questions/100021/when-transmembrane-proteins-destined-for-the-plasma-membrane-are-in-the-er-membr
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Question: <p>What is the most thermodynamically stable globular protein?</p>
<p>I am looking for a small (ideally less than 50kDa) soluble globular protein motif which folds easily/reliably and is known to be extremely stable and resistant to unfolding once folded. Also very resistant to proteolysis and degradation.</p>
<p>Any ideas?</p>
Answer:
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https://biology.stackexchange.com/questions/45566/what-is-the-most-stable-globular-protein
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Question: <p>I have been studying protein structure prediction algorithms. A lot of recent work uses something called the PSSM, the position-specific scoring matrix.</p>
<p>I think that what a PSSM does is to build a 2-D matrix of all possible residue pairs in a protein, then scores how likely it is that the two residues mutate in tandem. Co-evolution of two positions which are far from each other in the primary sequence are an indication that they are in contact. When one residue in a contact pair changes, that generally destabilizes the protein, and the other residue in the pair is under selective pressure to compensate. Knowing this gives you a good start on building the protein contact map.</p>
<p>Do I have that right?</p>
<p>If that is correct, then this technique of protein structure prediction depends on having many examples of proteins in a homologous family. You need nature to do a lot of sampling for you. And you need to dredge up massive numbers of homologs from genomics projects. I've read that creating the multiple sequence alignments for PSSM work is computationally intensive. If I understand the process correctly, I can see why.</p>
<p>My main question is: what can protein structure prediction models built using PSSM do, when there isn't a PSSM?</p>
<p>For example, the <a href="https://www.ncbi.nlm.nih.gov/pubmed/14631033" rel="nofollow noreferrer">Top7 protein is a fully-novel protein fold</a> that does not have any homologs in nature. It was created in 2003 using RosettaDesign software. Rosetta's protein structure prediction algorithms predate PSSM as far as I know. Sixteen years later, there are exactly six variants of Top7, all of which have been made in the laboratory. That hardly sounds like enough data for a statistically-valid PSSM, and in any case, the variants were not naturally selected.</p>
<p>If you don't have a PSSM, is it even possible to enter your sequence into a model that expects one?</p>
<p>Thanks for your input.</p>
Answer: <p>I think you are making PSSMs out to be much more sophisticated than they really are.</p>
<p>A PSSM is merely a scoring matrix — it gives position specific scores for each residue at a given location.</p>
<p>There is no explicit pairing of interacting residues though that does sound like an interesting approach ...</p>
<p>You can learn more about PSSMs from many sources on bioinformatics including <a href="https://www.ncbi.nlm.nih.gov/Class/Structure/pssm/mat_tutorial.html" rel="nofollow noreferrer">NCBI</a>.</p>
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https://biology.stackexchange.com/questions/85531/protein-structure-prediction-using-pssm-or-not
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Question: <p>I'm a computer scientist who is starting to dabble with biology. My eventual goal is to model different kinds of cells with a computer program. As of right now, I'm just trying to take some smaller steps.</p>
<p>First, I downloaded a complete human genome from <a href="http://hgdownload.cse.ucsc.edu/downloads.html#human" rel="noreferrer">http://hgdownload.cse.ucsc.edu/downloads.html#human</a> There is a FASTA file for each chromosome.</p>
<p>Then, I wrote a java program which can convert FASTA DNA sequences into the appropriate amino acid chain.</p>
<p>Next, I made my program look for the "start" code (ATG) and "stop" codes (TAA, TAG, TGA).</p>
<p>So, now I have sequences of amino acids which might theoretically end up folding into proteins. But, before I start diving into protein folding, I wanted to try to verify that the steps I took so far were done correctly. I looked up some important human genes in an online database and found their amino acid sequences. I then searched through my program's data for those sequences and confirmed that they were there. However, the gene was in a different base-pair location than the database said that it should be in.</p>
<p>This led me to some questions, which, so far I have been unable to answer and hopefully people here will be able to help shed some light.</p>
<ol>
<li><p>I know there are a lot of different publicly available genomes. Maybe the UCSC one that I downloaded is different from the one used by the gene database. How much does each genome vary from each other genome and in what ways do they vary?</p></li>
<li><p>In attempting to answer that first question, I was going to download a bunch of genomes from the 1000genomes website and do some comparisons, but I wasn't sure which files to download. Each of the files begins with either ERR or SRR and I'm not sure what that means. This is the folder I'm currently looking in <a href="ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/data/HG00239/sequence_read/" rel="noreferrer">ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/data/HG00239/sequence_read/</a></p></li>
<li><p>Lets say I'm trying to model a white blood cell. How do I know which parts of the genome get turned into proteins for that type of cell?</p></li>
</ol>
<p>Sorry if anything I said doesn't make sense. As I said, my expertise lies in programming, not biology/genetics.</p>
Answer: <p>No, your approach will not work, you are taking a very simplistic view of an extremely complex system. Some of the problems you are ignoring are:</p>
<ul>
<li><p>Genes (eukaryotic genes anyway) are <a href="https://en.wikipedia.org/wiki/RNA_splicing" rel="nofollow noreferrer">spliced </a>to produce mRNA, a process that removes <a href="https://en.wikipedia.org/wiki/Intron" rel="nofollow noreferrer">introns</a> and leaves only the <a href="https://en.wikipedia.org/wiki/Exons" rel="nofollow noreferrer">exons</a>. If you just translate the entire chromosome file you will get noise.</p>
</li>
<li><p>Splicing also changes the <a href="https://en.wikipedia.org/wiki/Reading_frame" rel="nofollow noreferrer">frame</a> a gene is read in, you don't mention frames at all in your question but you can't work with sequences unless you deal with them.</p>
</li>
<li><p>Many genes (most even, in some species) are <a href="https://en.wikipedia.org/wiki/Alternative_splicing" rel="nofollow noreferrer">alternatively spliced</a>. One gene can give rise to multiple protein sequences. Which one is produced at any one time can depend on a multitude of factors ranging from pure chance, through environmental conditions to the cell type where the gene is expressed.</p>
</li>
<li><p>Genes can be present on both strands of DNA and a gene on the + strand can overlap with a gene on the - strand. In some cases they can even overlap on the same strand (<a href="https://en.wikipedia.org/wiki/Nested_gene" rel="nofollow noreferrer">nested genes</a>). You need to check <em>both</em> strands for coding sequences.</p>
</li>
<li><p>You're assuming that all coding sequences start with ATG (most do, not all) and you seem to be assuming that an ATG always starts a coding sequence. A given gene can have dozens or hundreds of ATG codons, how can you know which one is used as a START codon?</p>
</li>
</ul>
<p>The process of identifying the parts of the genome that get translated into protein is not trivial. It is the subject of countless PhD theses, mine for example. There are many programs (gene predictors) that are designed specifically to detect genes in genomic sequences. Having spent many years working with them I can assure you that they're not something you can just whip up one afternoon. They tend to involve very complex models of coding vs. non-coding sequences and are way more sophisticated than simply looking for START and STOP codons. Trying to write one without knowing a <em>lot</em> more about biology than you seem to is just a waste of time.</p>
<p>Your specific questions are basically irrelevant because of the points mentioned above. Nevertheless, the answers are:</p>
<ol>
<li><p>They vary but not much. For well annotated genomes like the human one, the differences will be negligible. That is not why you have strange results though as I explained above.</p>
</li>
<li><p>All public FTP sites tend to have a README file that explains what the files provided are. You should read the relevant README from <a href="ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/" rel="nofollow noreferrer">ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/</a></p>
</li>
<li><p>Answering that question will get you a Nobel prize. There simply is no way of predicting what genes will be activated in a particular cell. We're not even <em>close</em> to that level of understanding of how a cell works but I can tell you that it will not depend on the sequence, you will never be able to predict whether a gene is active in a particular cell based on its DNA sequence alone. It will depend on various things including the gene's <a href="https://en.wikipedia.org/wiki/DNA_methylation" rel="nofollow noreferrer">methylation</a> state and is largely an emergent quality of the cell's complexity (think of various proteins interacting with one another, leading to the activation of a gene). The best you can do is get a list of genes that are known to be active from the literature.</p>
</li>
</ol>
<p>In summary, if you want to do something as complex as modelling a cell I suggest you first take the time and study some basic biology so you can understand the system you are trying to model a bit better. The cell is not only an extremely complex system that we don't fully understand yet, it is also not wholly deterministic and contains a lot of stochasticity that you seem to be ignoring completely.</p>
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https://biology.stackexchange.com/questions/19658/turning-publicly-available-genome-data-into-proteins
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Question: <p>What is the mechanism of bending of myosin head during the power stroke of the <a href="https://en.wikipedia.org/wiki/Sliding_filament_theory#The_sliding_filament_theory" rel="nofollow">cross-bridge cycle of the muscle contraction</a>? Does this have anything to do with the protein's 3-D structure i.e. folding of protein in space? I would prefer a physics explanation down to either the classical electrical dipole or quantum mechanical interaction amongst the proteins. I want to know what exactly produces the force that powers the bending. I am pretty sure that the force can only be electromagnetic. The question is how the force is manifested in the bending.
Any references for further reading are appreciated.</p>
Answer: <h1>Introduction:</h1>
<p>This is going to be quite a long answer. To have an introduction to the topic, you can have a look at articles from <a href="https://en.wikipedia.org/wiki/Myosin_head" rel="nofollow noreferrer">Wikipedia</a> and <a href="http://pdb101.rcsb.org/motm/18" rel="nofollow noreferrer">RCSB Protein Data Bank</a>.</p>
<p>The exact mechanism of physical interactions in myosin head during powerstroke cycle are not yet known. The only thing we definitely know about how release of P<sub>i</sub> from myosin causes conformational changes (and hence, force) in it, as given at <a href="http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_12%20Fall%202011.htm" rel="nofollow noreferrer">MJC</a> website is:</p>
<blockquote>
<p><a href="https://i.sstatic.net/roMM2.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/roMM2.jpg" alt="cross bridge cycle" /></a></p>
</blockquote>
<p>This is a very basic description of the powerstroke cycle, so we'll move ahead for more detailed explanation of physical interactions during the cycle.</p>
<h1>Details:</h1>
<p>I have found three theories regarding physical interactions during the powerstroke cycle. Let's discuss them one by one.</p>
<p><strong>THEORY 1:</strong> I could not find much explanation about physics of the cycle, the only thing I could find was at the <a href="http://www.jbc.org/content/289/18/12779.full" rel="nofollow noreferrer">JBC website</a> (emphasis mine):</p>
<blockquote>
<p>Mutation R759E in the myosin converter domain results in biochemical and biophysical defects as well as aberrant muscle structural and physiological properties. The central portion of the converter domain is encoded by exon 11e in indirect flight muscle (Fig., green), and the converter interfaces with the exon 9a-encoded relay domain (Fig., blue). Molecular modeling indicated that residues 508–511 in the relay loop are located near the converter residue 759 and defined weak and strong interactions of residues 509 and 511 during the rearrangements of the relay loop that are affiliated with the mechanochemical cycle. Furthermore, Ile508 can be cross-linked to Arg759 in <em>Dictyostelium</em> non-muscle myosin II when they are each substituted by cysteine. Therefore, we hypothesized that <strong>specific amino acids in the relay domain interact with converter residue 759</strong> and that second site mutations in the relay residues may suppress the defects associated with converter mutation R759E.</p>
</blockquote>
<p><a href="https://i.sstatic.net/VU2us.gif" rel="nofollow noreferrer"><img src="https://i.sstatic.net/VU2us.gif" alt="myosin" /></a></p>
<p>They couldn't give much explanation of the physics involved in it. Maybe this is because I missed it or because there hasn't been much research on this because <code>Dissecting the molecular mechanism of muscle myosin function in vivo has proved difficult.</code> as said at JBC website.</p>
<p><strong>THEORY 2:</strong> This theory, given at <a href="http://www.pnas.org/content/113/13/E1844.full" rel="nofollow noreferrer">PNAS</a>, provides a more detailed view of the physics, so we'll have a more thorough explanation of it:</p>
<blockquote>
<p>Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the <span class="math-container">$\beta$</span>-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a <span class="math-container">$\beta$</span>-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.</p>
</blockquote>
<p>Just so that you know what it is, let me add a few points about <a href="http://www.ks.uiuc.edu/Research/mdff/" rel="nofollow noreferrer">Molecular Dynamics Flexible Fitting</a>:</p>
<blockquote>
<p>The molecular dynamics flexible fitting (MDFF) method can be used to flexibly fit atomic structures into density maps. The method consists of adding external forces proportional to the gradient of the density map into a molecular dynamics (MD) simulation of the atomic structure. For examples of MDFF applications, visit the websites on <a href="http://www.ks.uiuc.edu/Research/ribosome" rel="nofollow noreferrer">Mechanisms of Protein Synthesis by the Ribosome</a>, <a href="http://www.ks.uiuc.edu/Research/translocon" rel="nofollow noreferrer">Dynamics of Protein Translocation</a>, <a href="http://www.ks.uiuc.edu/Research/STMV/" rel="nofollow noreferrer">Molecular Dynamics of Viruses</a>, and <a href="http://www.ks.uiuc.edu/Research/chromatophore" rel="nofollow noreferrer">Intrinsic Curvature Properties of Photosynthetic Proteins in Chromatophore</a>.</p>
</blockquote>
<p>Now, returning to the main point (results & discussion sections):</p>
<blockquote>
<p>The equilibrium and rates of transition between the Strong-ADP and the Rigor states vary greatly among different myosin isoforms and predominantly determine how long a myosin motor can remain bound to actin in the absence of load. This kinetic tuning must be achieved by structural differences in the regions that we have seen to change in our Strong-ADP structure as well as regions involved in stabilizing the lever arm position...Our structures show that the Loop 1 conformation alters in the transition from Strong-ADP to Rigor. Thus, different sequences likely favor one conformation over the other, or promote the transition from the ADP-bound conformation to the Rigor conformation, providing a structural basis for this kinetic tuning.</p>
<p>For myosins, such as myosin V, that function in a cell as two-headed, processive motors, the length of processive runs and the initiation of processive runs are both enhanced by “gating” of the heads. For a two-headed molecule with both heads simultaneously attached to actin, gating refers to the fact that a lead head is essentially stalled in an ADP state strongly bound to actin, until the rear head is detached from actin by binding MgATP. This gating is attributable to the strain dependence of MgADP release. Although we do not know whether some of the subdomains of myosin may be deformed by strain, our Strong-MgADP actomyosin structure clearly reveals that strain must prevent the rearrangement of the <span class="math-container">$\beta$</span>-sheet from the Strong-MgADP conformation to the Rigor conformation, based on the data presented in Results. Preventing this rearrangement is thus the basis of gating.</p>
</blockquote>
<p>For better understanding, you should also see <a href="http://movie-usa.glencoesoftware.com/video/10.1073/pnas.1516598113/video-8" rel="nofollow noreferrer">this</a> video (same website) which shows animation of different conformations of myosin in different stages of the cycle:</p>
<p><a href="http://movie-usa.glencoesoftware.com/video/10.1073/pnas.1516598113/video-8" rel="nofollow noreferrer" title="powerstroke animation"><img src="https://i.sstatic.net/XlgbUm.png" alt="powerstroke animation" /></a></p>
<p>The transitions are animated by direct morphing between the three structures Pre-Power Stroke (PPS), ADP state, and Rigor. Starting with the PPS conformation, myosin first rearranges to allow phosphate release without much change in its lever arm. Then, the first step of the powerstroke consists of a large swing of ∼58° of the lever arm toward the ADP state. The actin-binding cleft [between the U50 (blue) and L50 (white) subdomains] is closing for this transition, leading to the only state of the actomyosin cycle, which exhibits high affinity for both actin and the nucleotide. The second step of the powerstroke occurs upon ADP release and ends in the Rigor state after an additional lever arm swing of 9.5°.</p>
<p><strong>THEORY 3:</strong> This theory gives even more detailed, but a bit different from the previous one, view about how the energy, released by ATP hydrolysis, is stored in myosin head. It is called Rotation-Twist-Tilt (RTT) energy storage mechanism. See <a href="https://books.google.co.in/books?id=NfhsCQAAQBAJ&pg=PA161&lpg=PA161&dq=myosin%20convertor%20domain%20power%20stroke%20mechanism&source=bl&ots=5Ww33_WVcU&sig=3YSXQQj1FDleef1TuIIlZC4SJl4&hl=en&sa=X&ved=0ahUKEwixuZ2npozPAhVIGJQKHSXcDCcQ6AEIYTAJ#v=onepage&q=myosin%20convertor%20domain%20power%20stroke%20mechanism&f=false" rel="nofollow noreferrer">this</a>:</p>
<blockquote>
<p>According to the mechanism, ATP hydrolysis in the catalytic site rotates the top of the regulatory domain, which, being connected to the coiled coils of the S-2 region, causes twist between them to increase, and leads to the twist in the myosin head. Since, at the time of hydrolysis, the myosin molecule is not bound to the actin filament, the head is free to rotate and tilt. The increase in twist is instrumental in storing the energy of ATP hydrolysis, while the rotation and tilt of the myosin head brings it sufficiently close to the actin so as to form the actomyosin complex. Untwisting of the coiled coils and the subsequent untilting and constrained reversal of rotation of the head cause the power stroke. This strain decreases the energy of interaction between actin and myosin and thus enables ATP to dissociate myosin from actin. The system is now in such a state that after the next ATP hydrolysis event, the myosin head can bind to actin, and thus, a new contractile cycle can be initiated.</p>
</blockquote>
<p>The original paper of RTT mechanism (by Nath and Khurana, 2001) is available <a href="http://www.currentscience.ac.in/Downloads/article_id_081_01_0078_0082_0.pdf" rel="nofollow noreferrer">here</a>. Since the detailed mechanism is too long, I am not posting it here. You can read the full process, just remember to have a pen and paper with you!</p>
<p><a href="https://i.sstatic.net/kHsi1.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/kHsi1.png" alt="powerstroke cycle" /></a></p>
<h1>Conclusion:</h1>
<p>All of the above theories provide an in-depth view of the powerstroke cycle. However, I'd still conclude that the exact physics behind the powerstroke cycle is not yet fully known, only the transitions in conformation of myosin head have been observed and interpreted to some extent.</p>
<h2><em>References:</em></h2>
<ol>
<li><a href="https://en.wikipedia.org/wiki/Myosin_head" rel="nofollow noreferrer">Myosin head: Wikipedia</a></li>
<li><a href="http://pdb101.rcsb.org/motm/18" rel="nofollow noreferrer">Myosin: RCSB Protein Data Bank</a></li>
<li><a href="http://www.jbc.org/content/289/18/12779.full" rel="nofollow noreferrer">Journal of Biological Chemistry: Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function</a></li>
<li><a href="http://www.pnas.org/content/113/13/E1844.full" rel="nofollow noreferrer">Proceedings of the National Academy of Sciences of the United States of America: Force-producing ADP state of myosin bound to actin</a></li>
<li><a href="http://www.ks.uiuc.edu/Research/mdff/" rel="nofollow noreferrer">Theoretical and Computational Biophysics Group: Molecular Dynamics Flexible Fitting</a></li>
<li><a href="https://books.google.co.in/books?id=yd61229NHUgC&dq=how+phosphate+release+causes+change+in+shape+of+myosin+head&source=gbs_navlinks_s" rel="nofollow noreferrer">Cell Movements: From Molecules to Motility</a></li>
<li><a href="https://books.google.co.in/books?id=NfhsCQAAQBAJ&pg=PA161&lpg=PA161&dq=myosin%20convertor%20domain%20power%20stroke%20mechanism&source=bl&ots=5Ww33_WVcU&sig=3YSXQQj1FDleef1TuIIlZC4SJl4&hl=en&sa=X&ved=0ahUKEwixuZ2npozPAhVIGJQKHSXcDCcQ6AEIYTAJ#v=onepage&q=myosin%20convertor%20domain%20power%20stroke%20mechanism&f=false" rel="nofollow noreferrer">Molecular mechanisms of energy transduction in cells: Biotechnology in India II</a></li>
<li><a href="http://www.currentscience.ac.in/Downloads/article_id_081_01_0078_0082_0.pdf" rel="nofollow noreferrer">Molecular mechanism of the contractile cycle of muscle; Sunil Nath and Divya Khurana, March 2001</a></li>
</ol>
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https://biology.stackexchange.com/questions/51495/mechanism-of-myosin-head-bending-in-cross-bridge-cycle-power-stroke-phase
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Question: <p>I am interested in experimenting with folding simulations and algorithms for arbitrary sequences. I'm wondering if there is an easy way to convert an amino acid sequence into a PDB file for further simulation. To be clear I only want the primary protein structure.</p>
<p>If possible, I'd like to be able to characterize the bonds as well so that I can treat the molecule as a rigid body with rotational joints. Does anyone know something that can do this?</p>
Answer: <p>So to be clear, it sounds like you want the 'coil' or unfolded structure of a protein based on the sequence?</p>
<p>There are plenty of programs out there to do homology modelling, which is taking a sequence of unknown structure and modelling it onto one with a known structure. On the other hand, there are many libraries for analyzing existing structures. What you want is somehow in between these two.</p>
<p>I'll add some links here as I find them:</p>
<ul>
<li>There is a Lua library that might be useful : <a href="https://github.com/rob-miller/rFold" rel="nofollow noreferrer">https://github.com/rob-miller/rFold</a></li>
<li>Modeller has functions for this: <a href="https://salilab.org/modeller" rel="nofollow noreferrer">https://salilab.org/modeller</a> especially <a href="https://salilab.org/modeller/9.13/manual/node185.html" rel="nofollow noreferrer">this page</a></li>
<li><p>The Structural Bioinformatics Library : <a href="http://sbl.inria.fr/doc/index.html" rel="nofollow noreferrer">http://sbl.inria.fr/doc/index.html</a></p></li>
<li><p>The Rosetta library (as used in fold.it): <a href="https://www.rosettacommons.org/" rel="nofollow noreferrer">https://www.rosettacommons.org/</a></p></li>
</ul>
<p>I'm sure there are more, but it really depends on what programming language you want to use, or if you are implementing stuff yourself versus running some standalone software.</p>
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https://biology.stackexchange.com/questions/65972/building-a-pdb-file-from-amino-acid-sequence-of-non-folded-structure
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Question: <p>In studying the correlation of folded versus unfolded proteins and their impact on neuro-degeneration, it looks like improper phosphorylation in the chaperones (at least, in part) causes the mis-folding of proteins? If so, would it be possible to regulate proper phosphorylation so the UPR wouldn't initiate a reactionary cell death in important cells?</p>
Answer:
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https://biology.stackexchange.com/questions/34824/would-it-be-possible-to-regulate-proper-phosphorylation-so-the-upr-wouldnt-init
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Question: <p>If molecular chaperone proteins assist in the folding process of other proteins and misfolded proteins, can chaperone themselves misfold since they are also proteins? What would happen if chaperones misfolded? Can they misfold at all? Why or why not?</p>
Answer: <p>Chaperone proteins are still proteins and they can certainly misfold just like any other. If that happens, it will either be assisted by another chaperone and given time to fold successfully or it will be destroyed. If this is happening too often and the number of chaperones drop too low or the number of unfolded or incorrectly folded proteins becomes excessive<sup>*</sup>, the <a href="https://en.wikipedia.org/wiki/Unfolded_protein_response" rel="nofollow noreferrer">unfolded protein response</a> may be triggered and, if it does not resolve the issue and the cell remains stressed, the cell will undergo apoptosis and die.</p>
<p>Some chaperones, especially <a href="https://en.wikipedia.org/wiki/Heat_shock_protein" rel="nofollow noreferrer">heat-shock proteins</a>, may be more resistant to misfolding. This is true because they need to be able to withstand noxious conditions that denature other proteins. Not all chaperones are resistant to heat though and many are no more intrinsically resistant to denaturing.</p>
<p>* <sub> It's not uncommon for a "bad" batch of proteins to be created. This can happen with a transcription error during mRNA synthesis since each mRNA molecule is read by many ribosomes. This can naturally happen with chaperones as well.</sub></p>
|
https://biology.stackexchange.com/questions/81463/do-chaperone-proteins-misfold
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Question: <p>I used to think that a DNA clamp is a protein. But today I noticed it doesn't appear in <a href="https://upload.wikimedia.org/wikipedia/commons/8/8f/DNA_replication_en.svg" rel="nofollow noreferrer">this</a> picture. Then I went to it's Wikipedia page, where it was written: </p>
<blockquote>
<p>A DNA clamp, also known as a sliding clamp, is a protein fold that
serves as a processivity-promoting factor in DNA replication. As a
critical component of the DNA polymerase III holoenzyme, the clamp
protein binds DNA polymerase and prevents this enzyme from
dissociating from the template DNA strand. The clamp-polymerase
protein–protein interactions are stronger and more specific than the
direct interactions between the polymerase and the template DNA strand</p>
</blockquote>
<p>Which I find a little confusing. For now it seems to me like DNA clamp is a subunit of DNA Polymerase and it doesn't have any function by itself and it is not an actual protein but it binds to Polymerase and becomes functional. Can somebody please clarify this matter to me?! </p>
Answer: <p>Your partly right both ways. In a sense, the DNA clamp is a protein, in another sense, it's only part of a protein. What it actually is is what we call a protein sub-unit, which <em>is</em> a protein, but which binds with other protein sub-units to form complex proteins.</p>
<p>In order to understand this, you have to remember what a protein is. A protein is, in essence, a chain of amino acids. Those can be short chains, long chains, medium chains: but they're all proteins. Sometimes, a few short proteins (sub-units) will bind together to form one long protein (protein complex). This is what happens in the case of a DNA clamp.</p>
<p>Basically, the DNA clamp is (an) independent protein unit(s) which has (have) the ability to encircle the DNA strand and travel along it. They bind to the polymerase, which is why you don't see them on your diagram. Their function is, very simply, to make it possible for the polymerase to stay closely attached to the DNA stand.</p>
<hr>
<p>Sources:</p>
<ul>
<li><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691718/" rel="nofollow noreferrer">DNA Repair (Amst). 2009 May 1; 8(5): 570–578</a></li>
</ul>
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https://biology.stackexchange.com/questions/67489/what-is-a-dna-clamp-exactly
|
Question: <p>In <a href="http://en.wikipedia.org/wiki/Ribonuclease" rel="nofollow">this</a> link, it states:</p>
<blockquote>
<p>It is worth noting that all intracellular RNAs are protected from RNase activity by a number of strategies including 5' end capping, 3' end polyadenylation, and folding within an RNA protein complex (ribonucleoprotein particle or RNP).</p>
</blockquote>
<p>I do not understand how polyadenylation protects the intracellular RNA from ribonuclease. I always thought that polyadenylation was a way to mark RNA <em>for</em> degradation, not protect it from degradation. Perhaps it is speaking of protection from exoribonuclease only and not endoribonuclease?</p>
<p>Can someone explain this?</p>
Answer: <p>Actually, whether polyadenylation protects an mRNA or makes it susceptible to degradation depends on the organism. From <a href="http://www.sciencedirect.com/science/article/pii/S0092867402011376" rel="nofollow">Dreyfus and Régnier 2002</a>:</p>
<blockquote>
<p>In eukaryotes, poly(A) tails usually act as stabilizers of intact mRNAs, whereas in E. coli they serve to accelerate the destruction of fragments.</p>
</blockquote>
<p>This is due to different protection mechanisms in prokaryotes and eukaryotes:</p>
<blockquote>
<p>The Poly(A) Binding Protein (PABP) inhibits deadenylation in mammalian cell-free assays. Although poly(A) binding proteins exist in E. coli as well and although they can protect poly(A) tails against PNPase in vitro (Feng et al., 2001), they may be unable to exert a significant protection in vivo because poly(A) tails never grow long enough to enable them to bind.</p>
</blockquote>
<p>Important to note is that bacterial mRNA degradation usually starts <em>internally</em>, in contrast to eukaryotic mRNA degradation which happens (exonucleotically?):</p>
<blockquote>
<p>As noted above, another distinctive feature of bacteria is the fact that polyadenylation usually affects the degradation of fragments only, because the initial attack on most intact mRNAs is endonucleolytic.</p>
</blockquote>
<p>All of these quotes come from the review I referenced at the top of my answer, I recommend reading it as it's very comprehensive (and interesting).</p>
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https://biology.stackexchange.com/questions/34227/how-can-3-end-polyadenylation-protect-cellular-rna-from-ribonuclease-degridatio
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Question: <p>I am new to Western Blot analysis and I have recently done my first two. I am studying a phosphoprotein (a protein kinase) that can be both activated and inactivated via phosphorylation at a specific amino acid residue. I have labelled my membrane against the active and inactive forms of my protein of interest (using phosphospecific antibodies), as well as the total protein (using pan-specific antibodies).</p>
<p>I know that Western Blots are used to quantify the expression of a protein (i.e. how much of a specific protein there is in a sample). But I was wondering how they can be used to quantify the activity (e.g. how functional the protein is) of a protein, e.g. how can I measure/determine whether my protein is more active/inactive in my treatment samples vs. control?</p>
<p>For example, I have normalised the integrated density values for my active protein bands against the integrated density values for my total protein bands. Then I have calculated the fold change in protein level for my active protein in treatment samples relative to control.</p>
<p>If I have observed that the fold change for my active protein is reduced by 40% (as an example) in treatment samples relative to controls, would this be sufficient data to say that the activity of my protein of interest is reduced in treatment samples compared to control?</p>
Answer: <p>I see 3 parts to a complete answer:</p>
<ol>
<li><p>If you define the phosphorylated protein as active, and non-phosphorylated protein as inactive, and you have a total protein blot (as you say, via pan-specific antibodies) as you describe, <strong>then you have done everything correctly</strong>.</p>
</li>
<li><p>It's simple math from there on based on radioactivity or fluorescence or mass or whatever you use to quantify your band intensity. The 3 components should match up with the inactive protein amount (<em>active</em> + <em>inactive</em> = <em>total</em>). <strong>If this is consistent, you have additional evidence that you have done it correctly.</strong></p>
</li>
<li><p>However, <strong>here come the hidden assumptions</strong>: this is all assuming that (i) you are not experiencing significant protein loss during your preparation steps, that (ii) your antibodies are good (specific and yield the complete protein extract), that (iii) the blotting procedure is performed correctly, and <em>most importantly</em>, that (iv) your initial definition is valid, that the phosphorylated protein is truly the active form of the protein, and that the unphosphorylated counterpart is the inactive form. You see, this is not always the case, though with some (most?) kinases, it is. If it has not previously been demonstrated, one may raise the reasonable objection that your assumption is wrong. However, I take it that in your specific case, the kinase activation by phosphorylation is well understood.</p>
</li>
</ol>
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https://biology.stackexchange.com/questions/100182/can-western-blots-be-used-to-quantify-the-activity-of-a-protein
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Question: <p>I know about enhancers and the mechanism that lead them to increase the gene expression of their targets but I was wondering if similarly DNA repressors exist. I know about protein repressors but I am looking for some kind of anti(or reversed)-enhancer equivalent in the genome which would act like an enhancer but reduce gene expression.</p>
<p>I am aware of repressed/poised enhancers which would kind of be that process, even if not really a pure repression of the target gene but rather a "non-overexpression". I also know about insulators but again this would be a different mechanism.</p>
<p>Some intergenic region binding protein repressors which by a folding mechanism similar to enhancers would repress a gene perhaps?</p>
Answer: <p>Yes, these sequences exist and they are called "<a href="http://en.wikipedia.org/wiki/Silencer_%28DNA%29" rel="nofollow noreferrer">silencers</a>" (surprising, right?). There are different mechanisms by which this silencing of genes can happen.</p>
<p>In the "classical" way the silencer is bound by a transcription factor which either passively suppress the gene by hindering the binding of specific transcription factors or by actively preventing the assembly of the general transcription factors. See the figure from paper 1:</p>
<p><img src="https://i.sstatic.net/UyanS.png" alt="enter image description here"></p>
<p>Additionally there are non-classical negative regulatory element (NRE), which are usually elements upstream of the promoter which inhibit the binding regulatory proteins. NRE can also be enhancers depending on the proteins bound on them. Some NRE can induce a bend of the DNA, inhibiting the access to enhancer or promoter elements.</p>
<p>References:</p>
<ol>
<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/9512455" rel="nofollow noreferrer">Transcriptional control and the role of silencers in transcriptional
regulation in eukaryotes.</a></li>
<li><a href="http://bejerano.stanford.edu/readings/public/10_Intro_TxRegReview.pdf" rel="nofollow noreferrer">Transcriptional Regulatory Elements in the Human Genome</a></li>
</ol>
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https://biology.stackexchange.com/questions/30417/do-dna-repressors-exist
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Question: <p>Is there any protein database online where I could obtain a list of proteins ordered by the length of their chains / number of amino acids, starting from the shortest, as well as to see their amino acid sequences?</p>
<p>I'd like to start from the short & simple protein structures to see how their particular sequences of amino acids translate into their folding shape, and what those short chains are capable of doing in organisms. (I heard about the LILs, but they are artificially-generated and consist of just two amino acids, Lysine and Isoleucine, so not much variety to study in there :q )</p>
<p>But I'm quite new to these databases, they present a lot of details, but usually not the one I'm looking for, and one has to know what too look for first to get any meaningful information :q</p>
Answer: <p>You can find the data you need in the Protein Data Bank.</p>
<blockquote>
<p>Since 1971, the Protein Data Bank archive (PDB) has served as the
single repository of information about the 3D structures of proteins,
nucleic acids, and complex assemblies.</p>
<p>The Worldwide PDB (wwPDB) organization manages the PDB archive and
ensures that the PDB is freely and publicly available to the global
community.</p>
</blockquote>
<p>Each of the PDB member organisations provides the same data with a different interface:</p>
<ul>
<li><a href="https://www.ebi.ac.uk/pdbe/" rel="nofollow noreferrer">PDBe</a>,</li>
<li><a href="https://pdbj.org/" rel="nofollow noreferrer">PDBj</a>,</li>
<li><a href="https://www.rcsb.org/" rel="nofollow noreferrer">RCSB</a>.</li>
</ul>
<p>In general, you either query the database through one of the web interfaces, or you download all the data and search it locally. In this case, the RCSB website has the option you asked about (sorting by the residue count):</p>
<p><a href="https://i.sstatic.net/o1wlz.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/o1wlz.png" alt="RCSB search screenshot"></a></p>
<p>Alternatively, you could download:
<a href="ftp://ftp.wwpdb.org/pub/pdb/derived_data/pdb_seqres.txt" rel="nofollow noreferrer">ftp://ftp.wwpdb.org/pub/pdb/derived_data/pdb_seqres.txt</a>
and parse it and sort it as you wish.
This file has sequences of all chains in the PDB entries.</p>
<p>But since your goal is to find the relationship between the sequence and the folded structure, you should probably start from reading about the protein folding problem and about methods used in protein structure prediction.</p>
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https://biology.stackexchange.com/questions/76943/list-of-proteins-by-number-of-amino-acids-chain-length
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Question: <p>Trying to get a better understanding of the process of DNA to proteins.</p>
<p>So when we have a gene, it is read from the 5' to 3' end, only translating the exons to mRNAs. A single gene can have multiple exons, and use alternative splicing to create different transcripts. A transcript may result in mRNA (not necessarily right? Also other kinds of RNA?) which is translated to amino acids. Finally folded to a functional protein.</p>
<p>My question here, are there different transcripts (say in different genes) that encode the same protein?</p>
Answer:
|
https://biology.stackexchange.com/questions/36679/multiple-transcripts-encoding-one-protein
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Question: <p>Say I transduce a nucleic acid sequence using a viral vector that encodes a protein having at least one disulfide linkage. For simplicity, let’s assume that there are only two cysteines in the protein and the side chains of these cysteines are close together when the protein is folded with the cysteines unlinked (reduced/thiol). Also assume that the cell being transduced is a eukaryotic cell (mouse or human). What is the chance that this disulfide will be linked (oxidized) when expressed by the cell? Does this happen pretty reliably? What might it depend on?</p>
Answer: <p>I am not an expert on expression of genes from viral vectors, but as I requested the poster to clarify his question I feel an obligation to provide at least a partial answer. </p>
<p>The first question to address is whether the mRNA for the protein of interest is expressed in the same way as normal cell proteins. In the case of the <a href="https://en.wikipedia.org/wiki/Viral_vector#Lentiviruses" rel="nofollow noreferrer">lentovirus vectors</a> which are integrated into the genome there is no reason to think otherwise.</p>
<p><em>In this case the factors determining the formation of disulphide bonds would be the same as for any similar normal cell protein translated from mRNA.</em> </p>
<p>In summary:</p>
<ol>
<li><p>Only secreted proteins have disulphide bonds.</p></li>
<li><p>In eukaryotes these protein are synthesised on ribosomes on the endoplasmic reticulum, which obviously must be present in the cell in which one is trying to express the protein (i.e. it must be capable of secreting proteins). One assumes that if one is restoring a function found in normal cells this will be so.</p></li>
<li><p>Oxidation of sulphydryl groups occurs in the intercisternal space before secretion. It involves a number of proteins and there are reduction and chaperone systems to deal with inappropriate oxidation, which occurs to a greater or lesser extent. A recent freely-accessible account of this topic can be found in a <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536336/pdf/cshperspect-ERT-a013219.pdf" rel="nofollow noreferrer">Cold Spring Harbor perspective</a> by Neil Bulleid in 2012.</p></li>
<li><p>The (small?) proportion of incorrectly oxidised proteins that are not recycled are presumably removed by the cellular mechanisms that degrade aberrant proteins.</p></li>
</ol>
<p>It would not seem possible to answer in a literal manner the question what the numerical ‘chance’ is of correct oxidation, but this would not seem to be pertinent. The question of interest is whether the protein would be oxidised to the correct native structure with the same efficiency as in normal circumstances. There seems no reason to think it would not.</p>
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https://biology.stackexchange.com/questions/87837/formation-of-disulfide-bonds-in-protein-expressed-after-transduction
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Question: <p>I'm curious about how protein structures are defined in general, but in particular, I'm wondering how a target structure can be specified without knowing the amino acid sequence. </p>
<p>For example, in protein design (or from what I gather on the wikipedia page <a href="http://en.wikipedia.org/wiki/Protein_design" rel="nofollow">http://en.wikipedia.org/wiki/Protein_designs</a>), scientists design a 3D structure that will perform a particular function. They then try to come up with an amino acid sequence that will fold properly into that structure. </p>
<p>How can a 3D structure be defined if no amino acid sequence (and hence no molecular geometry) has been determined?</p>
Answer:
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https://biology.stackexchange.com/questions/28536/protein-design-target-structure-specification
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Question: <p>I initially thought that a domain was a specific part of a protein, with it given tertiary structure, to which a given molecule is able to bind. (I think I recall phrases such as "the haem binding domain of protein X..." being used in lectures?)</p>
<p>Having consulted Wikipedia on protein domains, I see the definition is a bit more subtle:</p>
<blockquote>
<p>A protein domain is a conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. </p>
</blockquote>
<p>I can understand this, however the reason why I started questioning what a 'domain' actually refers to was because if it's use in reference to Sda in control of endosporulation of bacteria. In my lecture notes, it is stated that "KinA is bound and destabilised by Sda, a DnaA target,"</p>
<p>From this I was under the impression that Sda is a protein (although to the best of my knowledge DnaA only binds DNA, so I do not know why Sda would be target of DnaA. Anyhow,) on the other hand Wikipedia states "the protein domain Sda is short for suppressor of dnaA or otherwise known as sporulation inhibitor A.", which seems to me to suggest that Sda is a part of DnaA whose modifications allow DnaA suppression?</p>
<p>Also, on Wikipedia, it is later written "Sda protein domain is a checkpoint which prevents the formation of spores." How can a protein, if Sda is one, be a checkpoint?</p>
<p>On the other hand my lecture notes later talk about the regulation of Sda levels, so it again is referred to as a separate protein!</p>
<p>I would very much appreciate if someone could explain what 'domain' means in this context.</p>
Answer: <p><strong>Short answer</strong></p>
<p>Wikipedia has unnecessarily made an already confusing situation much worse. Sda is its own protein, it is regulated by DnaA, and it prevents sporulation.</p>
<p><strong>Full answer</strong></p>
<p>I don't think your misunderstanding is based on the meaning of 'domain' - the definition you give sounds quite reasonable - but rather the use of the term domain with respect to Sda and the naming of Sda itself, which I agree is quite confusing and made much worse by Wikipedia in this case.</p>
<p>Sda is a protein that inhibits KinA activity (Burkholder et al., 2001) and is a completely separate protein from DnaA. I don't see where you jumped from your notes to the idea that Sda is a domain of DnaA except for from Wikipedia: I don't know why Wikipedia thinks Sda is a protein domain, it is a protein. It is a very small protein, so perhaps it would be appropriate to think of it all as one domain, but <em>there is not a single result for "Sda protein domain" on Google Scholar</em> - no one else is using this terminology. A full Google search only returns a few (~150) results, most of which are direct copies/translations from Wikipedia, or Wikipedia is a direct copy of them. Wikipedia can be useful, but if it starts to cause you confusion, don't assume it is correct.</p>
<p>The phrasing "KinA is bound and destabilised by Sda, a DnaA target" means that Sda binds KinA and destabilizes it, and notes that Sda is a target of DnaA; since DnaA is a transcription factor, you can infer that this means that DnaA controls expression of Sda in some way. </p>
<p>In biology a checkpoint just refers to a place that a process can be halted/arrested, often in reference to the cell cycle. Sda is considered a checkpoint because when Sda is expressed, it prevents cells from sporulating by indirectly preventing activation of Spo0A (Burkholder et al., 2001).</p>
<p>Another source of confusion is that the name "suppressor of dnaA" on Wikipedia is <strong>WRONG</strong>, but further, molecular biologists sometimes have an impenetrable way of naming things, owing to the complexity of control of gene expression and the circuitous way that new proteins are identified and understood.</p>
<p>The correct name for <em>sda</em> is "suppressor of <em>dnaA1</em>". <em>dnaA1</em> is NOT DnaA - <em>dnaA1</em> is a MUTANT ALLELE of DnaA that results in cells that are completely unable to sporulate. But! <em>sda</em> is also NOT Sda: <em>sda</em> is a MUTANT ALLELE that produces non-functional Sda (the protein).</p>
<p>I'll quote from the title of the table in Burkholder et al:</p>
<blockquote>
<p><em>sda Mutations Suppress the Sporulation Defect of dnaA1 Mutants</em></p>
</blockquote>
<p>Or, stated again, in <em>dnaA1</em> mutants, there is a defect in DnaA which prevents sporulation. The researchers searched for mutations that would reverse this effect. They found one, and named it sda, because it reverses (suppresses) the effect of the mutation they were studying. The <em>dnaA1</em> mutant's effects on sporulation seem to be caused by an increased expression of Sda, which is why the sda mutant reverses the effect.</p>
<p>References</p>
<hr>
<p>Burkholder, W. F., Kurtser, I., & Grossman, A. D. (2001). Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis. Cell, 104(2), 269-279.</p>
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https://biology.stackexchange.com/questions/58701/is-sda-a-protein-or-is-it-a-protein-domain-of-dnaa
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Question: <p>The DNA is read 3'->5' (and RNA synthesized 5'->3'). But due to the DNA strands having to be complementary, it seems to me that the origin can only appear on the correct side of the ORF on one of the strands. And even if it were possible to synthesise RNA from the other strand, due to the directionailty of the strands/RNA the RNA transcript would then code for the protein in reverse. Surely the reverse synthesis of a protein, even if containing all of the same amino acids, would lead to dfferent folding and thus only on eof the proteins is correct?</p>
<p>Now that I am considering the origin, it seems likely to me that the strand on which the origin is dictates which strand acts as the RNA template (perhaps both starnds can, however for a given GENE the RNA is always synthesised from the same strand.</p>
<p>Is this the case? I have read this post: <a href="https://biology.stackexchange.com/questions/1966/majority-of-transcripts-are-from-sense-strand">Majority of transcripts are from sense strand?</a> however I found it slightly confusing. I think what the answerer said is similar to what I have suggested here, however I am not quite sure. Would greatly appreciate some input.</p>
Answer:
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https://biology.stackexchange.com/questions/58438/are-rna-transcripts-always-synthesised-from-the-same-dna-strand
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Question: <p>So throughout my education and research career I have been taught that all enzymes are proteins. This makes sense when you consider enzyme denaturing and folding/shape etc. However, I was recently told by a biology professor that in fact, not all enzymes are proteins -- and he alluded to the RNA world hypothesis and rRNA's. </p>
<p>Could someone explain further how exactly an rRNA (or similar) could be (or not be) considered an enzyme, and whether or not all enzymes are proteins etc?</p>
<p>Thank you!</p>
Answer: <p><strong>Disclaimer</strong></p>
<p>I voted to close this question as it struck me that the by giving the poster the magic word ‘ribozyme’ he could easily verify for himself that the answer to the question in his title was “No”. However, as this question received answers that I find incorrect or misleading, I provide my own answer to put the record straight.</p>
<p><strong>History and Terminology</strong></p>
<p>Although I would not necessarily regard entries in Wikipedia as authoritative, scientific dates are not particularly contentious, so I refer the reader to the entries on <a href="https://en.wikipedia.org/wiki/Enzyme" rel="nofollow noreferrer">Enzyme</a> and <a href="https://en.wikipedia.org/wiki/Protein#History_and_etymology" rel="nofollow noreferrer">Protein</a> for the following:</p>
<ul>
<li>The first enzyme to be discovered is said to have been diastase, in 1833, although the opposition of Pasteur to the idea of metabolic activity outside of living things delayed the acknowledgement of this. The word <em>enzyme</em> was coined in 1877 from the Greek, meaning “in yeast”. The catalytic activity of the enzymes of fermentation was studied intensively in the late 19th and early 20th century, culminating in Buchner’s Nobel Prize in Chemistry in 1907.</li>
<li>Although proteins had been recognized in the 18th century, and named by Berzelius in 1838, their chemistry as polypeptides was not established until 1902, and it was not until 1926 that Sumner established that urease was a protein.</li>
</ul>
<p>Hence, the word enzyme and the concept of its activity was introduced long before the chemical nature of most enzymes as proteins was established, and therefore the poster is correct in <em>not</em> including protein in the definition of enzyme. It is also correct to talk about the later discovery of enzymes that are RNA rather than protein, despite the fact that the latter confounded a general belief that had held for over a half century. Indeed, RNA enzymes are referred to by a special designation — <strong>ribozymes</strong> — just as most members of genus <em>Cygnus</em> are referred to as swans, but <em>Cygnus atratus</em> is usually referred to as <strong>black</strong> swans.</p>
<p><strong>RNA enzymes (ribozymes)</strong></p>
<p>A perfect example of an RNA enzyme would be one that was naturally <em>active in the absence of protein</em> and showed <em>catalytic activity against a distinct substrate</em>. As of 2002, when the topic was <a href="https://www.nature.com/articles/418222a" rel="nofollow noreferrer">reviewed by Thomas Cech</a> — who, together with Sidney Altman, received the Nobel Prize in Chemistry for the discovery of the first ribozyme in 1962 — there were no such natural examples.</p>
<ul>
<li>The type of catalytic RNA discovered by Cech — the self-splicing introns of <em>Tetrahymena</em> are completely free of protein, but act on the pre-mRNA that contains them.</li>
<li>Another type of catalytic RNA, exemplified by the ribonuclease-P discovered by Altman, is active against a substrate distinct from itself — a precursor tRNA — contains protein as well as RNA. However if the protein is removed from RNase-P under appropriate conditions the RNA component retains catalytic activity.</li>
</ul>
<p>Despite these limitations — and the fact that the only reactions catalysed are cleavage and ligation of RNA — I would have cited these as clear examples of RNA enzymes if I had been the professor to whom the poster addressed his question. Ribosomal RNA probably has catalytic activity, but is less clear-cut, as I explain below.</p>
<p><strong>The ‘RNA World’ and RNA catalysis of other types of reaction</strong></p>
<p>One of the results of the discovery was catalytic RNA in the early 60s was that it gave flesh to an idea that resolved some of the problems of the early evolution of biochemical processes — especially the interdependence of protein and nucleic acid. This was an ‘RNA World’ in which RNA served both as genetic material (before DNA evolved) and the enzymes for performing biochemical reactions, including that of protein synthesis. However the only reactions catalysed by the contemporary RNA enzymes that had been characterized were the cleavage or ligation of RNA itself.</p>
<p>One could argue that other catalytic reactions were taken over by proteins when they emerged, as they were more efficient etc., however the question remained as to whether RNA had the potential to catalyse such reactions. One step in that direction was to artificially select oligoribonucleotides (aptamers) on the basis of their ability to bind particular small molecules. There have also been <a href="https://rnajournal.cshlp.org/content/5/11/1482.full.pdf" rel="nofollow noreferrer">reports</a> that a small synthetic RNA can catalyse the amino acylation of AMP-activated tRNA for phe.</p>
<p><strong>The peptidyl transferase activity of rRNA</strong></p>
<p>It is in the context of what I have written above that the possible catalytic activity of rRNA should be viewed.</p>
<ul>
<li>The reaction involved is that of the peptidyl transferase (below). There are no other examples of RNA catalysing reactions other than RNA hydrolysis and ligation</li>
<li>The implications for the ‘RNA World’ hypothesis (which is <em>not</em> universally accepted) are such that — although I personally favour it (and have lectured to students on it in relation to ribosomes) — one must be very careful not to allow this to cloud one’s scientific judgement.</li>
</ul>
<p><a href="https://i.sstatic.net/zvQBp.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/zvQBp.png" alt="Peptidyl Transferase" /></a></p>
<p>The evidence for the peptidyl transferase (PT) activity of the large ribosomal RNA (23S rRNA in <em>E.coli</em>, 28S rRNA in <em>H.sapiens</em>) is as follows:</p>
<ul>
<li>Repeated attempts to associate particular ribosomal proteins with PT activity failed and many individual ribosomal proteins could be deleted from <em>E.coli</em> ribosomes without abolishing the PT activity</li>
<li>Affinity labels of substrates of the PT reaction tended to label RNA rather than protein.</li>
<li>The PT centre has been located in the three-dimensional structure of 23S rRNA by crystalization with analogue of the reaction intermediate. No proteins are in the vicinity of this.</li>
</ul>
<p>This argues strongly that the PT activity of the ribosome lies in the 23S rRNA — but cannot be regarded as a rigorous proof. Not surprisingly, no protein-free RNA or fragment thereof has been shown to be able to catalyse the PT-reaction between the small substrate analogues that can be used for this.</p>
<p>I happen, myself, to believe that the ribosome is a ribozyme — for the little that it is worth — but I would never put forward the ribosome as an example of a ribozyme, when so much better ones exist.</p>
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https://biology.stackexchange.com/questions/79633/are-all-enzymes-proteins
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Question: <p>It may sound silly, but it appeared to me as a theoretical possibility; not a practical fear. </p>
<p>On a healthy and correctly folded protein if cooking process is applied (that includes heating and mixing with various other things), is there by any chance a possibility to develop a prion? </p>
Answer: <p>Just my speculation here. Prions seem to need some sort of template to guide their folding as well as the cooperation of chaperone proteins. <a href="https://en.wikipedia.org/wiki/Prion" rel="nofollow noreferrer">https://en.wikipedia.org/wiki/Prion</a> I would suspect that denaturing, and potential re-folding, of proteins during cooking would be rather random. So even if one, or very few, copies of a potential prion happened to be produced, the high temperatures, rapid pace of denaturation and the lack of chaperone proteins would not allow the formation of meaningful amount of potential prions to cause disease. Add to that the doubtful chance a prion would be produced in the first place, due to the lack of the usual physiological conditions and participants, feel free to eat cooked food!</p>
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https://biology.stackexchange.com/questions/56019/could-the-denaturation-of-protein-during-cooking-could-generate-a-prion-by-any
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Question: <p>We work with a membrane protein system where measuring the affinity between the enzyme and the upstream activating protein has been difficult, and when measured in detergent solution, it is almost 100 fold lesser (ie ~100nM) whereas the EC50 in an enzymatic assay using vesicles in ~1-2nM. Would it be reasonable to say that the "real" affinity is ~1nM than 100s of nM based on the biochemical assay?</p>
<p>Alternately, is there a documented system where a huge discrepancy exists between measurements from direct binding and biochemical assays?</p>
Answer: <p>You can certainly get massive differences between EC<sub>50</sub> and affinity. This is especially true for cell-based assays and membrane protein systems. </p>
<p>The reason why is because the appropriate time scales to achieve binding equilibrium (hrs for nM affinity, days for picomolar, feptomolar affinity according to back of the envelope calculations) may be and likely will be different from the appropriate time scales of activation, endocytosis, degradation, etc. Furthermore, you can have avidity effects that would not be apparent in a dilute membrane assay compared to a cell's surface. It could very much be that the dominant terms in mass transport change depending on the geometry and antigen density and while I'm not entirely sure bout vesicles vs. emulsions it is true for cancer models. <a href="http://www.ncbi.nlm.nih.gov/pubmed/12649189" rel="nofollow noreferrer">Theoretical analysis of antibody targeting of tumor spheroids: importance of dosage for penetration, and affinity for retention.
</a></p>
<p><strong>Case study number I:</strong> hGH:hGHR interactions have been extensively studied and high affinity hGH have been produced. However, none of the variants showed improved EC<sub>50</sub>. It turns out that the binding was primarily influenced by the off-rates which were so slow, that affecting those values did little to the kinetics of the system. <a href="http://pubs.acs.org/doi/abs/10.1021/bi9817008" rel="nofollow noreferrer">Growth Hormone Binding Affinity for Its Receptor Surpasses the Requirements for Cellular Activity</a></p>
<p><strong>Case study number II:</strong> CEA-antibodies have been affinity matured to have pM activity compared to the wild-type nM affinity. However, the antibody had minimal effect on tumor uptake. As CEA gets endocytosed on the order of 30 minutes, all of the antibody was simply getting endocytosed rather than acting as an inhibitor. <a href="http://peds.oxfordjournals.org/content/17/4/293.abstract" rel="nofollow noreferrer">Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37°C</a>.</p>
<p>(edit)
I realized that this answer is more applicable to IC<sub>50</sub>s vs. the EC<sub>50</sub> experiments described above. However, many of the points remains. The Km between a substrate and its enzyme is a thermodynamic property where as the EC<sub>50</sub> also has dynamic factors that may influence its measurement.</p>
<p>I would also add that simply adjusting the pH 2 units may result in a 2 orders of magnitude change in activity.</p>
<p>(edit2)
A further point to make about enzymatic assays is that from an observation of Michaelis-Menten kinetics, the K<sub>M</sub> of the reaction isn't the K<sub>D</sub> of the enzyme but alternatively the effective binding constant.</p>
<p>Recall that:</p>
<p><img src="https://i.sstatic.net/Vb9Lm.png" alt="enter image description here"></p>
<p>If there is a non-trivial k<sub>cat</sub> comparable to the dissociation rate k<sub>r</sub>, the enzyme will have an effective half maximal concentration that is significantly higher than the K<sub>d</sub>. There are multiple ways how your assay can result in the discrepancies that you're seeing. The first would your strategy in measuring your EC<sub>50</sub>. If one is an enzymatic readout and the other is a binding readout, you would naturally have very different results. Alternatively, the enzyme in the detergent system may be catalytically more active than the vesicle system resulting in a higher effective K<sub>M</sub>. Again, the pH thing.</p>
<p>Finally, there is always that awkward moment when you realized that you might accidentally have measured a titration curve rather than a binding curve.</p>
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https://biology.stackexchange.com/questions/3147/is-the-ec50-of-an-activating-protein-for-an-enzyme-a-good-indicator-for-the-bind
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Question: <p>Bortezomib is an anti-cancer drug that inhibits the proteasomes of cancer cells, allowing proteins that stop cell growth to fold and perform their function.</p>
<p>However, wouldn't bortezomib also affect the proteasomes of normal cells? If the proteasomes of normal cells are inhibited, the major factor in the regulation of unfolded and damaged proteins is gone. The resulting protein aggregate would lead to cell damage and diseases such as cancer, Alzheimer's and Parkinson's. </p>
<p>What is stopping bortezomib from affecting normal cells? Does it?</p>
Answer: <p>Proteasome inhibitors do affect normal cells to some extent, but the whole point of using them as cancer treatments is that (some) cancer cells are far more sensitive to proteasome inhibitors than are normal cells. For example, multiple myeloma cells (the first clinical targets of Bortezomib) that overproduce immunoglobulins are dependent on proteasome degradation to eliminate them and are therefore <a href="http://www.mdpi.com/1424-8247/10/2/40" rel="noreferrer">sensitive to Bortezomib</a>. There are many other aspects of cancer cells that may make them more dependent on proteasome; see for instance </p>
<p><a href="http://www.sciencedirect.com/science/article/pii/S0891584916310942" rel="noreferrer">Proteasome inhibitors in cancer therapy: Treatment regimen and peripheral neuropathy as a side effect</a> for some examples.</p>
<p>But of course there are effects on normal cells as well, such as the peripheral neuropathy mentioned in that review. </p>
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https://biology.stackexchange.com/questions/60065/if-bortezomib-a-cancer-drug-inhibits-cell-proteasomes-wouldnt-resulting-prot
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Question: <p>The protein isoforms I am interested in comparing appear as distinct bands on the gel I have already run. I have an Excel sheet with optical density measurements I obtained using ImageJ; it looks something like this:</p>
<pre><code>Lane Iso1 Iso2 GAPDH
1 149.06 194.646 893.08
2 832.654 494.473 148.335
3 49.998 490.539 147.361
4 29.347 208.53 120.652
</code></pre>
<p>For the other proteins I've analyzed so far, I've been computing fold-change relative to my loading control, GAPDH, with the following equation:</p>
<pre><code>Fold-Change = Log2(Protein/GAPDH)
</code></pre>
<p>I'm not sure how best to compare my isoforms to each other and considering the following two equations:</p>
<pre><code>1. Fold-Change = Log2[(Iso2/GAPDH)/(Iso1/GAPDH)]
2. Fold-Change = Log2(Iso2/Iso1)/GAPDH
</code></pre>
<p>I've already computed both of these values for all my samples, graphed them and found that the resulting graphs look pretty different. <strong>Which of the two equations do you think is a better way to compare the relative quantities of these proteins?</strong></p>
<p>Alternative equations are also welcome.</p>
Answer: <p>In my opinion you should use this formula:</p>
<p>$$ \frac{\text{log}_2(\text{Iso}_1/\text{Iso}_2)}{\text{log}_2(\text{GAPDH})} $$</p>
<p>This will normalize the relative fold differences between the isoforms with the loading control- GAPDH.</p>
<p>Since both numerator and denominator are log transformed they are in comparable domains unlike the formula-2 that you mention in your question. This is just a modified formula-2.</p>
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https://biology.stackexchange.com/questions/21931/what-equation-to-compare-protein-isoforms-in-a-western-blot
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Question: <p>Well first I am not in the field of Biology or Medical Sciences. Since these days we are waiting scientists to tell us if the Indian variant of SARS Cov 2 is more transmissible than the original virus, so these two questions come to my mind:</p>
<ol>
<li>Do scientists confirm increase in transmissibility based on empirical data?</li>
<li>Can scientists know how transmissible the variant is based on how well the spike protein binds to receptors of the cell, for example by doing a simulation of the 3D folded shape of the spike based on its code in the genome?</li>
</ol>
Answer: <p><a href="https://ncceh.ca/documents/evidence-review/basics-sars-cov-2-transmission" rel="nofollow noreferrer">Generally based on (1)</a>. To quote the website:</p>
<blockquote>
<p>The variants that are cause for most concern may:</p>
</blockquote>
<ul>
<li>spread more quickly,</li>
<li>evade natural or vaccine-related immunity,</li>
<li>cause more severe disease,</li>
<li>evade detection by available tests, or</li>
<li>are less responsive to treatment.</li>
</ul>
<p>If you look at the features they say they look for in variants, the one they mention first is "spread more quickly" - the only way to tell that is to look at the genetics and how often the variant is turning up in a relative to a previous time-point. Bullet point 2 can only be seen by testing - do people who have had an infection, get infected again? Bullet point 3 - look at the severity of disease in patients.</p>
<p>I am sure by now that you get the drift; surveillance is the answer - all empirical data.</p>
<p>(2) may or may not help, all that does is tell you the potential affinity for a receptor, which is only one part of the transmissibility. In fact, we generally need to wait for the emergence of a variant (no point chasing each one; every infection will have some form of variant in some of the virions produced - they exist as a <a href="https://www.sciencemediacentre.org/expert-reaction-to-the-new-variant-of-sars-cov-2/" rel="nofollow noreferrer">quasispecies</a>), before modelling will take place. In addition, modelling is actually a pretty poor method of working out whether the virus will bind to a receptor. Check out <a href="https://www.pnas.org/content/117/36/22311" rel="nofollow noreferrer">this paper</a> and see that many of the Mustelid family have poor binding by modelling, but in reality they actually get infected easily by SARS-CoV-2.</p>
<p>Many other factors come into viral transmission, such as viral titre in the patient, number of viral particles being shed in each droplet/aerosol, survivability of the particles in the air/droplet/aerosol etc.</p>
<p>Here's a <a href="https://www.pnas.org/content/113/15/4170" rel="nofollow noreferrer">nice paper from PNAS</a> that covers the biological features of transmissibility based on a statistical/modeling analysis. While this doesn't specifically address SARS-CoV-2, it does cover a bunch of the features that are looked at for increased transmission in viruses overall.</p>
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https://biology.stackexchange.com/questions/100602/virus-variant-transmissibility-empirical-data-or-spike-protein-shape
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Question: <p>From Lewin's Genes (11th edition, page 515):</p>
<blockquote>
<p>The only bacterial RNA polymerases for which high-resolution crystal structures have been solved, however, are from two thermophilic bacterial species, <em>Thermus aquaticus</em> and <em>Thermus thermophilus</em>.</p>
</blockquote>
<p>Is this just a matter of economy? I mean, as X-ray crystallography is exceedingly expensive and complex, did scientists consider enough to resolve the RNA polymerase structure of one species and deduce those belonging to the rest by homology-based folding prediction?</p>
<p>Or is it because thermophilic proteins are inherently more stable and thus easier to crystalize?</p>
Answer: <p>I think both of your suggestions have some merit: (1) <a href="https://www.nature.com/articles/261725a0" rel="nofollow noreferrer">it appears that <em>Thermus</em> RNAPs are more stable and easier to crystallize</a> (2) RNAP is highly conserved and so it is not critical to crystallize it from every bacterial species. </p>
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https://biology.stackexchange.com/questions/82555/why-do-the-most-structurally-well-characterized-bacterial-rna-polymerases-belong
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Question: <p>I have been measuring my protein solutions' concentrations by diluting them in water 20 fold with a final volume of 100 uL and then measuring the absorbances of these solutions in 96 well plates with plate reader. I don't remember having any problem up until today.</p>
<p>I used 20 mM phosphate buffer instead of water to dilute them today and measured the absorbances at 280 nm repeatedly three times and the absorbance for one of the solutions went up from 0.043 to 0.068 (absorbance of 20 mM phosphate buffer is 0.030 at 280 nM with same volume); I stopped measuring after third one but it would probably go higher as I measured until I hit a plateau.</p>
<p>I measured absorbances of two proteins and only one of them went up that much, other one went up from 0.071 to 0.088; if this were to be concentration dependent I would expect the second solution go even higher but it didn't happen.</p>
<p>I know there may be differences in UV absorbances if protein is folded or unfolded; would it be that dramatic? What is the reason for that increase? I will be grateful for an explanation and a practical solution to the problem.</p>
<p>NOTE: I increased the total volume to 200 uL by simply adding 100 uL of 20 mM phosphate buffer into all wells and the signal increase slowed down a lot; there is still some increase though with 0.001-0.003 increments in each measurement.</p>
Answer: <p>It looks like your protein concentrations are right on the limit of detection of the spectrophotometer, and changing the diluent buffer changed their concentrations. The samples may not have been thoroughly mixed after dilution and before measurement, so the varying measurements may simply be the solution coming to equilibrium. Temperature can also affect absorbance, so you should verify that your samples have equilibrated before drawing any conclusions. If the absorbance of your phosphate buffer is 0.03, I'd try to keep the sample absorbances above 0.075 or higher to avoid getting too close to the limit of detection. Also, make sure your buffer isn't too old or contaminated with something which could be affecting its absorbance characteristics.</p>
<p>I would suggest taking one or two protein samples and doing a dilution series (1:1, 1:5, 1:10, 1:20) in a large-ish volume (say 400 ul each, if you can spare it), vortex briefly to mix well, then measure triplicates of each dilution on your reader, along with appropriate blanks (buffer only). You will see differences between each measurement, but it should be quite small, depending on the accuracy and precision of your instrument. </p>
<p>Measured values will not be exactly the same from measurement to measurement, and it would take a lot more than three repetitions to determine if there was an actual drift trend occurring. Measure your sample plate every 5 minutes for an hour and plot the values (don't just eyeball them) to see if the machine may need to be serviced.</p>
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https://biology.stackexchange.com/questions/7124/why-is-absorbance-at-280-nm-for-protein-solution-going-up-when-i-measure-repeate
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Question: <p>I was wondering about the shapes assumed by mRNA. I have read some sources quoting that <a href="https://www.quora.com/What-shapes-do-the-mRNA-tRNA-and-rRNA-have" rel="nofollow noreferrer">it is linear (quora, so not very reliable)</a> and also a source that says a <a href="https://www.nature.com/scitable/definition/hairpin-loop-mrna-314/" rel="nofollow noreferrer">hairpin shape is common (nature, so I would rather go with this one)</a>.</p>
<p>I was also wondering what additional factors or features ensure the mRNA has this shape. For instance, the first source says that the mRNA 'is linear so that the ribosomes can bind to it'. But, clearly, all that is required for this is that the ribosome binding site is exposed. In that case, the remainder of the mRNA may either spontaneously unravel as it is being translated, or there may be something slightly more specific taking place catalysed by the ribosome, or there may be an additional enzyme that associates and catalyses the unfolding of the mRNA as it is being translated.</p>
<p>The thing is, I just find it difficult to belive that the mRNA would not fold up on itself, but on the other hand if it did, perhaps it would be too stable and would not be degraded sufficiently easily. We would have accmulation of mRNA in the cell, unless there was a protein that specifically degraded it. </p>
<p>My only other hypothesis, if it truly is the case that the mRNA does not fold up, is that it is down to the DNA in the nucleaus being 'too dense' to permit the mRNA folding in the nucleus, and once it exits via a nuclear pore it is immediately being translated and forming a polyribosome complex. So then it can no longer fold.</p>
<p>I do not think that the DNA content in the nucleus is sufficiently dense for steric effects like this. I have seen electron micrograph images in a book (Molecular Biology of the Cell) of mRNA exiting a nuclear pore. It seems quite coiled up. Unfortunately, I have been unable to find a clear representative image online or the associated paper. But perhaps the mRNA can coil up in the nucleus, but is straightened out as it is pulled throught the nuclear pore. Proteins and factors associated with the nuclear pore may catalyse the breaking of the hydrogen bonds.</p>
<p>Any details on this would be much appreciated.</p>
Answer: <p>Unfortunately it is often taught that mRNA is linear, but this is not true at all. Nucleotides within mRNA can form intra-molecular hydrogen bonds with other nucleotides, creating secondary structures, interactions between these secondary structures results in tertiary structures. mRNAs have highly variable secondary structures, primarily due to differences in the sequence of the ORF, so there is not a consensus structure adopted by mRNAs. However, codon degeneracy is important in producing more ordered and stable mRNA secondary structures in ORFs, resulting in increased mRNA half-life. Nucleotides in positions one and two in codons are the most conserved, whereas the third nucleotide position is the least conserved and has elevated GC content, thus making the greatest contribution to the stability of the secondary structure. The 5’ UTR typically contains lots of secondary structures, which may help in modulation of translation initiation, whereas 3’ UTRs contain fewer secondary structure, resulting in structural instability around binding sites for proteins and miRNAs, enhancing miRNA and protein binding. The start and stop codons are less frequently base-paired than the rest of the mRNA, suggesting they exist in local secondary structures which lead to efficient initiation and termination of translation. However, the sequence immediately downstream of the stop codon has a high GC content and an in increase in frequency of base pairing. It has been hypothesised that this strong secondary structure is present to interrupt post-termination ribosomal scanning. Whilst increasing stability of mRNA increases half-life, low secondary structures around binding sites within the mRNA suggests that thermodynamic stability of mRNA is optimised and not-minimised in all scenarios. </p>
<p>With regards to translation initiation there is quite a lot of cool work on dynamic secondary RNA structures for regulating translation initiation, this work primarily revolves around riboregulators and riboswitches. I won't go into detail about them here, but for a great paper on riboregulators see: Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–39 (2014)</p>
<p>References:</p>
<p>Comparative analysis of orthologous eukaryotic mRNAs: potential hidden functional signals. Nucleic Acids Res. 32, 1774–82 (2004).Shabalina, S. A., Ogurtsov, A. Y., Rogozin, I. B., Koonin, E. V & Lipman, D. J.</p>
<p>Duan, J. & Antezana, M. A. Mammalian Mutation Pressure, Synonymous Codon Choice, and mRNA Degradation. J. Mol. Evol. 57, 694–701 (2003).</p>
<p>Shabalina, S. A., Ogurtsov, A. Y. & Spiridonov, N. A. A periodic pattern of mRNA secondary structure created by the genetic code. Nucleic Acids Res. 34, 2428–2437 (2006).</p>
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https://biology.stackexchange.com/questions/88054/the-shape-of-mrna
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Question: <p><a href="http://en.wikipedia.org/wiki/Hidden_Markov_model">HMM</a> alignment tools like hhpred excel at finding subtle homologues of folded proteins that simpler scoring techniques (such those used in BLAST algorithms) would miss.</p>
<p>I am only looking at a small (20AA) sequence and it is helical throughout. </p>
<p>Is hhpred still likely to pick up on subtle similarities in the basic secondary structure as it would in a folded protein sequence, or would simpler alignment be just as appropriate?</p>
Answer: <p><a href="http://www.cbs.dtu.dk/services/TMHMM/" rel="nofollow">TMHMM</a> is a very good standard on predicting the TMHs in the first place, so it stands to reason that predicting homologues using this approach is completely viable.</p>
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https://biology.stackexchange.com/questions/28350/is-using-hidden-markov-models-to-find-homologues-sensible-in-abstract-short-seq
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Question: <p>I was reading about prions and many sources say something to this effect:</p>
<p>"Prions may propagate by transmitting their misfolded protein state: When a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the misfolded prion form. In this way, the prion acts as a template to guide the misfolding of more proteins into prion form." (Wikipedia)</p>
<p>How can/does a prion cause another protein to change its shape to match the prion's? </p>
Answer: <p>It is thought that infectious prions exist as clusters forming a crystalline structure. When a protein with the same primary structure is encountered but with a different tertiary structure, the normal protein undergoes a conformational change in order to integrate into the cluster. Presumably there are molecular forces involved that induce the conformational change.</p>
<p><a href="http://www.rsc.org/chemistryworld/Issues/2005/October/prions.asp" rel="nofollow">http://www.rsc.org/chemistryworld/Issues/2005/October/prions.asp</a></p>
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https://biology.stackexchange.com/questions/45629/how-do-prions-transmit-their-conformation-to-other-proteins
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Question: <p>Membrane insertion of transmembrane proteins typically requires highly hydrophobic alpha helixes at the N-terminus, N-terminal signal peptides, tail anchors, or a combination of the three.</p>
<p>Byun, H., Gou, Y., Zook, A., Lozano, M., & Dudley, J. (n.d). ERAD and how viruses exploit it. Frontiers In Microbiology, 5</p>
<p>These processes occur co-translationally and are mediated by the SEC translocon and associated factors (especially GPI anchor transferases for tail-anchored proteins). However, my undergraduate research is focused on a viral protein which appears to be translated in association with SRP and co-translationally translocated to the ER by the translocon, but is not anchored to the membrane during translocation. This was shown (by others) by purification of the ER-associated fraction of the protein and centrifugation showing the majority pelleted with the dense, soluble fraction. A small fraction pelleted with the low density membrane-associated fraction, and my research further suggests that this membrane-associated fraction is important for its escape from the ER.</p>
<p>The protein I am studying functions in the cytoplasm, not in the ER or in the secretory pathway, and therefore its escape from the ER is essential for its function. The typical ERAD pathway for soluble proteins involves retrotranslocation through the Sec61 translocon channel (the same channel involved in co-translational translocation, but with different associated factors during ERAD), but this would result in unfolding of the protein prior to its retrotranslocation to the cytosol. On the other hand, the ERAD pathway for membrane proteins (the "dislocation pathway") appears to dislocate folded membrane proteins while retaining most or all of their tertiary/quaternary structure (previous citation and below).</p>
<p>Avci, D., & Lemberg, M. K. (2015). Clipping or Extracting: Two Ways to Membrane Protein Degradation. Trends In Cell Biology, (10), 611. doi:10.1016/j.tcb.2015.07.003</p>
<p>Knowing that the protein is not co-translationally inserted into the membrane because only a small fraction is membrane associated, and suspecting that the small membrane-associated fraction is important for its escape from the ER because the dislocation pathway allows folded protein to exit the ER, my question is:</p>
<p>How could the retrotranslocation pathway for soluble proteins be disrupted such that it would (infrequently) result in membrane insertion of this protein <strong>at an early step in retrotranslocation-coupled unfolding, such that the folded protein could follow the dislocation pathway into the cytoplasm</strong>?</p>
<p>I would appreciate any examples of similar processes in viruses or in eukaryotes, but any speculation on possible mechanisms - based on understanding of ERAD but lacking supporting examples - would also be greatly appreciated.</p>
<p>As this is unpublished research, I can't be too specific about what virus or even what model system I'm working in, but if there is additional information that would help, I'll be happy to provide it - if I can.</p>
<p>One final piece of information that is also relevant is that I suspect the protein's transmembrane or membrane-anchored domain is very close to its C-terminus. Another important question that this raises is <strong>whether retrotranslocation exclusively proceeds by feeding the N-terminus of a protein into the Sec61 channel, or if the C-terminus can be fed directly into the channel?</strong> If only the N-terminus could be fed into the channel, this would suggest most of the protein would have to be unfolded before the C-terminal domain became associated with the channel and was able to become membrane-associated, which voids the main benefit of the dislocation pathway; maintaining the folded state of the protein.</p>
<p>If somebody can answer only this question regarding the translocon (and provide some relevant reading), I would be incredibly grateful because it would suggest further literature search to help support the mechanism I am proposing.</p>
<p>Thank you!</p>
Answer:
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https://biology.stackexchange.com/questions/45666/are-there-well-studied-examples-of-erad-mediated-membrane-insertion-especially
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Question: <p>A prion is an abnormally folded protein that is capable of causing otherwise normal proteins to also misfold and become prions. They are responsible for causing diseases such as Kuru and Creutzfeldt–Jakob disease. These are both diseases of the brain. Are there any non-brain proteins that also have corresponding prions that can cause disease, or do prion diseases only affect the brain?</p>
Answer: <p>Yes, malformed prion proteins can affect tissues outside of the brain.</p>
<p>Via: <a href="https://www.merckmanuals.com/home/brain,-spinal-cord,-and-nerve-disorders/prion-diseases/overview-of-prion-diseases" rel="nofollow noreferrer">https://www.merckmanuals.com/home/brain,-spinal-cord,-and-nerve-disorders/prion-diseases/overview-of-prion-diseases</a></p>
<blockquote>
<p>Another familial prion disease has been recently discovered. It differs from other prion diseases because it causes diarrhea and affects nerves throughout the body years before symptoms of brain malfunction develop. It is described as prion disease associated with diarrhea and autonomic neuropathy.</p>
</blockquote>
<p>Via: <a href="https://www.merckmanuals.com/home/brain,-spinal-cord,-and-nerve-disorders/prion-diseases/prion-disease-associated-with-diarrhea-and-autonomic-neuropathy" rel="nofollow noreferrer">https://www.merckmanuals.com/home/brain,-spinal-cord,-and-nerve-disorders/prion-diseases/prion-disease-associated-with-diarrhea-and-autonomic-neuropathy</a></p>
<blockquote>
<p>Symptoms begin when people are in their 30s. People have persistent watery diarrhea and bloating. They may lose weight. Because the nerves that control body processes are affected, people may not be able to pass urine (called urinary retention) or may lose control of their bladder (urinary incontinence). Their blood pressure may drop when they stand up, causing them to feel dizzy or light-headed (called orthostatic hypotension). People may lose sensation in their feet. Later, when people are in their 40s or 50s, mental function deteriorates, and seizures may occur.</p>
</blockquote>
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https://biology.stackexchange.com/questions/88012/are-there-any-proteins-not-found-in-the-brain-that-are-affected-by-prions
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Question: <p>I am part of an undergraduate research group and we are trying to produce a protease in an E.coli gene circuit. We are not sure where to place the His tag in our circuit. The sequence is
...OmpA(gene)->linker->his tag->protease->Terminator->res site
Should the His tag be in this position, before the linker, or after the protease? OmpA is a outer membrane protein that will allow a protein to be taken to, and linked to, the outer membrane of the E.coli. Our fear is that the protease will not fold correctly and will be nonfunctional. </p>
Answer:
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https://biology.stackexchange.com/questions/35489/his-tag-location-in-gene-circuit
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Question: <p>Can ccr4-not enter a cell? To stimulate mrna de-adenylation</p>
<p>Bartel and colleagues found a 1000 fold variation in mRNA degradation rates: <a href="https://wi.mit.edu/news/be-long-lived-or-short-lived" rel="nofollow noreferrer">https://wi.mit.edu/news/be-long-lived-or-short-lived</a></p>
<p>In addition Iwasaki and colleagues found spike protein induced in the nucleus from mRNA vaccine up to 700 days after vaccination. <a href="https://news.yale.edu/2025/02/19/immune-markers-post-vaccination-syndrome-indicate-future-research-directions" rel="nofollow noreferrer">https://news.yale.edu/2025/02/19/immune-markers-post-vaccination-syndrome-indicate-future-research-directions</a></p>
<p>A method to import ccr4-not into long-COVID spike producing cells may help degrade mRNA in those who still have it in there. If not, the existing mRNA vaccine would continue to generate spike proteins - perpetuating long COVID.</p>
Answer: <p>The multi-protein Ccr4-Not complex participates in poly(A) tail removal from mRNA which results in decay of the message. The messenger RNA stability function of this complex helps control gene expression regulation at the molecular level.</p>
<p>Can Ccr4-Not enter a cell?</p>
<p>Eukaryotic cells contain Ccr4-Not inside their cytoplasm as a group of interacting proteins which make up the complex. Since it exists inside cells already Ccr4-Not does not have to enter because it directly interacts with mRNA molecules within the cell.</p>
<p>How does the Ccr4-Not complex initiate the degradation process that shortens mRNA poly(A) tails?</p>
<p>Within the Ccr4-Not complex exist deadenylase enzymes including Ccr4 and Caf1 that shorten the poly(A) tail attached to mRNA.</p>
<p>The shortening of poly(A) tail leads to mRNA instability which triggers its destruction process.</p>
<p>Such a process serves essential gene regulation purposes by enabling effective removal of unnecessary or faulty mRNAs.</p>
<p>As a primary component in post-transcriptional regulation the Ccr4-Not complex enables cellular protein production and enables environmental adaptation through poly(A) tail removal.</p>
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https://biology.stackexchange.com/questions/116367/can-ccr4-not-enter-a-cell
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Question: <p>From what I read, it seems the current situation is like this: latest cutting-edge lab techniques get to an accuracy of around 90%. For example, they put a protein under an electron microscope or something, and from the images obtained they achieve a precision of 90%. Now Deepmind features an AI that has demonstrated in contests that it can also virtually fold proteins to 90% accuracy. Therefore, the problem is kind of solved.</p>
<p>Please correct me if my interpretation is wrong. But if it's correct, then a big question remains: how the hell can they know if a configuration is 100% true? Even the best technology can only reach 90%, so where do they get that 100% for comparison?</p>
Answer:
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https://biology.stackexchange.com/questions/97372/alphafold-how-do-they-determine-the-100-accuracy-threshold
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Question: <p>We did 4 experiments to compare the amount of certain proteins in treated and untreated cells. Each experiment was done separately. Because of the high cost of experiment, we were able to perform only one pair (one treated and one untreated) sample for each experiment. We want to see which proteins are differentially expressed (minimum 1.5 fold up/down). </p>
<p>First approach: We have compared protein levels of all 4 treated (as a group) with the 4 untreated (as a second group). There is of course variability between all experiments, because of the nature of the cells. We have a list of the proteins that are differentially expressed as a result of the treatment, however this list is not very long. </p>
<p>My question is (second approach): Can we compare the proteins levels pairwise for each experiment (treated vs its respective control) and make 4 corresponding lists, and then compare these four list using a statistical tool and find which proteins are consistently up- or down-regulated? DO you think that these two different approach will generate different lists of the affected proteins?</p>
Answer: <p>If I understand you did a treatment to some cells and compared them with non-treated ones. Instead of running the four experiments at the same time, you did one treated and one untreated at a time. Then you did proteomics for each sample. Is the treatment the same in all four experiments? </p>
<p>Edit after the further comments of the OP: So, since the four treated samples are the same and the four untreated also (same conditions except from the treatment and the treatment is the same), then the way to go is as what your collaborator did. </p>
<p>Identification and quantification of the detected proteins is one thing and every sample of the four are replicates. Comparison between the two conditions is another thing.
The software he uses for ID can combine the same samples and already perform the statistical analysis, so the list you have now has higher credibility in terms of the proteins it includes and their levels for your cells when they are treated or not.</p>
<p>Using only one of your replicates, although it might give you different number of detected proteins or different amounts of each, has lower credibility, because it's only one sample out of four. In plain words, the presence or absence or the amount of a protein might be an artifact or insignificant.</p>
<p>What has to be clear is that the comparison between treated and untreated conditions is done after you have received the statistically correct list of detected proteins.</p>
<p>Thus, and in accordance to what I had said before the edit, if you take the list for each treated sample and compare them with any of the lists of the untreated ones, it will lead to conclusions that won't be as statistically significant as when you combine all treated together and all untreated together (as your colleague did). In plain words, your conclusions will have a higher chance to be wrong.</p>
<p>Every statistical analysis you do yourself for each sample should eventually lead to a similar consensus as your colleague got using the statistical analysis of his software.</p>
<p>As a sidenote, considering how many types of proteins are in a cell, now that you have a quite short list might not be a bad thing at all and you can proceed by:</p>
<ol>
<li>Concluding that the treatment had minor effect in the proteins that you expected it would affect (if they are not present in your lists as significant different)</li>
<li>Trying to understand, identify and hypothesize on the role of the proteins that made it in your comparison threshold, as they have a much higher probability of being indeed different between the two conditions.</li>
</ol>
<p>Splitting the samples in your analysis might have a point if the conditions of the experiment were not exactly the same or the treatment level is different etc. In that case you could split the samples accordingly, but that would definitely reduce your certainty level for your conclusions.</p>
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https://biology.stackexchange.com/questions/53669/proteome-patterns-between-treated-and-control-cells
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Question: <p>In a related post on Biology-SE the following insightful comment was made:</p>
<blockquote>
<p>The advantage of DNA shuffling over introducing single mutations is
that you have to screen fewer mutants and the activity/stability of
the protein could be improved several hundred fold more.</p>
</blockquote>
<p>Is there a mathematical argument as to why DNA Shuffling should be more efficient than introducing point mutations?</p>
<p>Even supposing each DNA Shuffling / Point Mutation step is equally "expensive" (and in reality DNA shuffling seems much more work) they are both generating one variant per-step, right? So why is the variant in one case grossly better than the other?</p>
<p>Related Post: <a href="https://biology.stackexchange.com/questions/45188/directed-evolution-point-mutation-vs-insertion-deletion-vs-shuffling">Directed evolution: Point mutation vs Insertion-Deletion vs Shuffling</a></p>
Answer: <p>You should read <a href="http://www.nature.com/nature/journal/v391/n6664/fig_tab/391288a0_F4.html" rel="nofollow noreferrer">this paper</a>. Here is the gist of what you are interested in:</p>
<blockquote>
<p>Because most point mutations are deleterious or neutral, the random point mutation rate must be low and the accumulation of beneficial mutations and the evolution of a desired function is relatively slow in such experiments. For example, the evolution of a fucosidase from a galactosidase required five rounds of shuffling and screening before a >10-fold improvement in activity was detected4. Naturally occurring homologous sequences are pre-enriched for 'functional diversity' because deleterious variants have been selected against over billions of years of evolution...</p>
<p>Although shuffling of a single gene creates a library of genes that differ by only a few point mutations1, 2, 3, 4, 5, 6, the block-exchange nature of family shuffling creates chimaeras that differ in many positions. For example, in previous work a single beta-lactamase gene was shuffled for three cycles, yielding only four amino-acid mutations3, whereas a single cycle of family shuffling of the four cephalosporinases resulted in a mutant enzyme which differs by 102 amino acids from the Citrobacter enzyme, by 142 amino acids from the Enterobacter enzyme, by 181 amino acid from the Klebsiella enzyme and by 196 amino acids from the Yersinia enzyme. The increased sequence diversity of the library members obtained by family shuffling results in a 'sparse sampling' of a much greater portion of sequence space15, the theoretical collection of all possible sequences of equal length, ordered by similarity (Fig. 4). Selection from 'sparse libraries' allows rapid identification of the most promising areas within an extended sequence landscape (a multidimensional graph of sequence space versus function)</p>
</blockquote>
<p><a href="https://i.sstatic.net/0Yte1.gif" rel="nofollow noreferrer"><img src="https://i.sstatic.net/0Yte1.gif" alt="enter image description here" /></a></p>
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https://biology.stackexchange.com/questions/45572/why-is-dna-shuffling-more-efficient-than-point-mutation
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Question: <p>The tail of RNA polymerase II is flexible, not folded into a fixed structure , but does each repeat have more "rigid" structure (i.e. fold into a structure that has less rotation freedom inside a repeat)? If not, then how can the proteins bind some repeat with high specificity? (you can't make lock and key with soft material)</p>
Answer:
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https://biology.stackexchange.com/questions/97034/how-does-rna-polymerase-ii-ctd-bind-to-the-rna-modification-proteins-if-the-tail
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Question: <p>I am currently reading a textbook (Molecular Biology of the Cell, 6th ed), and this problem on p. 170 is driving me crazy. I read through the solution given in this book but I couldn’t understand it after all.</p>
<p>Here’s the question:</p>
<p>Titin, which has a molecular weight of about 3 × 10<sup>6</sup>, is the largest polypeptide yet described. Titin molecules extend from muscle thick filaments to the Z disc; they are thought to act as springs to keep the thick filaments centered in the sarcomere. Titin is composed of a large number of repeated immunoglobulin (Ig) sequences of 89 amino acids, each of which is folded into a domain about 4 nm in length (Figure Q3–2A). You suspect that the springlike behavior of titin is caused by the sequential unfolding (and refolding) of individual Ig domains. You test this hypothesis using the atomic force microscope, which allows you to pick up one end of a protein molecule and pull with an accurately measured force. For a fragment of titin containing seven repeats of the Ig domain, this experiment gives the sawtooth force-versus-extension curve shown in Figure Q3–2B. If the experiment is repeated in a solution of 8 M urea (a protein denaturant), the peaks disappear and the measured extension becomes much longer for a given force. If the experiment is repeated after the protein has been cross-linked by treatment with glutaraldehyde, once again the peaks disappear but the extension becomes much smaller for a given force.</p>
<p><a href="https://i.sstatic.net/L0M5I.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/L0M5I.jpg" alt="enter image description here" /></a></p>
<p>A. Are the data consistent with your hypothesis that titin’s springlike behavior is due to the sequential unfolding of individual Ig domains? Explain your reasoning.</p>
<p>B. Is the extension for each putative domain-unfolding event the magnitude you would expect? (In an extended polypeptide chain, amino acids are spaced at intervals of 0.34 nm.)</p>
<p>C. Why is each successive peak in Figure Q3–2B a little higher than the one before?</p>
<p>D. Why does the force collapse so abruptly after each peak?</p>
<p>The solution:</p>
<p>A. These data are consistent with the hypothesis that the springlike behavior of titin is due to the sequential unfolding of Ig domains. First, the fragment contained seven Ig domains and there are seven peaks in the force-versus-extension curve. In addition, the peaks themselves are what you might expect for sequential unfolding. Second, in the presence of a protein denaturant, conditions under which the domains will already be unfolded, the peaks disappear and the extension per unit force increases. Third, when the domains are cross-linked, and therefore unable to unfold, the peaks disappear and extension per unit force decreases.</p>
<p>B. The spacing between peaks, about 25 nm, is almost exactly what you would calculate for the sequential unfolding of Ig domains. The folded domain occupies 4 nm, but when unfolded, its 89 amino acids would stretch to about 30 nm (89 × 0.34 nm), a change of 26 nm.</p>
<p>C. The existence of separate, discrete peaks means that each domain unfolds when a characteristic force is applied, implying that each domain has a defined stability. The collection of domains unfolds in order from least stable to most stable. Thus, it takes a little more force each time to unfold the next domain.</p>
<p>D. The sudden collapse of the force at each unfolding event reflects an important principle of protein unfolding; namely, its cooperativity. Proteins tend to unfold in an all-or-none fashion (see Problem 3–35). A small number of hydrogen bonds are crucial for holding the folded domain together (Figure 3–39). The breaking of these bonds triggers cooperative unfolding.</p>
<p>If so, I think the graph should look somewhat similar to this:</p>
<p><a href="https://i.sstatic.net/8QIn6.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/8QIn6.jpg" alt="Graph 1" /></a></p>
<p>or this:</p>
<p><a href="https://i.sstatic.net/HKkpw.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/HKkpw.jpg" alt="Graph 2" /></a></p>
<p>*I didn’t scale the graph because I don’t know the exact figure.</p>
<p>Since the protein unfolds in an all-or-none fashion, it won’t extend until it is unfolded, but it will extend gradually as it unfolds.</p>
<p>As it takes a little more force each time to unfold the next domain, the constant portion of the graph should be lengthened as the domains progressively unfold.</p>
<p>In both graphs, the extended lengths are always the same, but in graph 2 the domains get harder and harder to unfold so they require extra input of energy each time they unfold. (Thus the slope of the diagonal portions of the graph needs to be less steepened.)</p>
<p>I don’t know which one is correct, so I drew both.</p>
<p>The question is, why is my answer different from that of the textbook? Is there something wrong with my reasoning?</p>
<p>Any idea or answer is appreciated!</p>
Answer: <p>When an Ig domain unfolds, its length increases, so the force required to stretch the whole protein to a given length will decrease. Your diagrams don't account for this decrease in force.</p>
<p>You can think of the domains like wire coiled into the shape of a stereotypical spring when they are folded, and a straight line when they are unfolded. Stretching the coiled spring to the length of the straight wire will take a lot of force. Obviously no force is needed to keep the straight wire at its natural length. (This isn't true of unfolded proteins, but the <a href="https://en.wikipedia.org/wiki/Entropic_force#Polymers" rel="nofollow noreferrer">entropic force</a> is small compared to the elastic resistance from the folded domains)</p>
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https://biology.stackexchange.com/questions/111502/the-springlike-behavior-of-titin-problem
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Question: <p>Someone claimed that the dinosaurs could only live because back then athmospheric density was around 650 kg/m³, providing some buyoncy, this claim is <a href="https://skeptics.stackexchange.com/questions/19073/what-was-the-density-of-prehistoric-earth-atmosphere-dinosaurs-buoyancy-theory/20516">checked on Skeptics SE</a>. The claim itself is beside the point now. A simple calculation shows us that an atmsphere with this density would be <a href="http://en.wikipedia.org/wiki/Supercritical_fluid" rel="nofollow noreferrer">supercritical</a> in underwise normal conditions, the pressure would be increased by a factor of roughly 520. See my <a href="https://skeptics.stackexchange.com/a/20516/16410">answer to the skeptics question</a> for a bit more explanation. I would say that a supercritical air atmosphere rules out life for several reasons:</p>
<ul>
<li>The high effusity of the fluid may mean that cell membranes don't work</li>
<li>Proteins fold differently under high pressure, requiring specific adaptions</li>
<li>If the partial pressure of oxygen is increased proportionally, the atmosphere is highly toxic</li>
<li>If the partial pressure of oxygen remains at current levels, it is a trace gas and reactions relying on oxygen may run into kinetic problems (There may be a sweet spot between the last two issues)</li>
</ul>
<p>Anyway, those are just my hunches, I never heard of experiments about life in supercritical fluids and I'm no biologist so my hunches may be totally wrong. My question is: <strong>Can we rule out life in supercritical fluids;</strong> if not <strong>can we rule out air breathing, multi cellular animal life?</strong><br>
Or has any life been shown to thrive in a supercritical fluid?</p>
<p>To stress again, the part with buoyant dinosaurs was given for background (and amusement), I'm only asking here to check my hunch that suprecriticality rules out life.</p>
Answer:
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https://biology.stackexchange.com/questions/17191/is-life-in-a-supercritical-fluid-possible
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Question: <p>The following commentator <a href="https://www.physics.leidenuniv.nl/index.php?id=11573&news=889&type=LION&ln=EN" rel="nofollow noreferrer">writes</a>:</p>
<blockquote>
<p>Mechanical cues
Since the mid 80s it has been hypothesized that there is a second layer of information on top of the genetic code: DNA’s mechanical properties. Each of our cells contains two meters of DNA molecules, so these molecules need to be wrapped up tightly to fit inside a single cell. The way in which DNA is folded, determines how the letters are read out, and therefore which proteins are actually made. In each organ, only relevant parts of the genetic information are read, based on how the DNA is folded. The theory goes that mechanical cues within the DNA structures determine how DNA prefers to fold.</p>
</blockquote>
<p>My question is: <strong>Is there enough evidence in this paper that there is a second (mechanical) layer of information in DNA?</strong></p>
Answer: <p>Biologists already know that transcription can be regulated by winding or unwinding DNA. (This paper might be informative:<a href="http://www.cell.com/trends/parasitology/fulltext/S1471-4922(16)30226-4" rel="nofollow noreferrer">http://www.cell.com/trends/parasitology/fulltext/S1471-4922(16)30226-4</a>)</p>
<p>But this is hardly the only method of gene regulation.</p>
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https://biology.stackexchange.com/questions/69409/is-there-enough-evidence-in-this-paper-that-there-is-a-second-mechanical-layer
|
Question: <p>I need to optimize a transfection protocol to transiently express a plasmid encoding a chimera of eyfp attached to the c term of a Golgi apparatus signaling molecule) in hela cells and hepg2 cells and get as high expression as I can get.</p>
<p>I need enough protein for about 100 wb's. I've seeded hela cells to 12 150mm dishes. And the same with the hepg2. I've prepped mg's of plasmid.</p>
<p>I'm using invitrogens <strong>original</strong> lipofectamine reagent; not the 2000, or ltx or any of that stuff.</p>
<p>Any suggestions on a protocol here for hela and hepg2 plasmid dna and lipofectamine mediated transfection on such a large plate size. </p>
<p>I figure just scaling up should work but there's some pitfalls here. Look at this table for example when you go from 60 to 100 mm you about double the area, so you also about double the culture volume. However when you go from 100 to 150mm you triple the area, but only increase the culture volume two fold about. This is making my scale-up calculations for transfection look really off:</p>
<p><img src="https://i.sstatic.net/xRetR.jpg" alt="culture volumes and areas"></p>
Answer: <p>So, for every row on the dish protocol, the entire process is scaled up a discrete value. In the case of 100mm to 150mm, the values are scaled up by about 2.76 across all values:</p>
<p>(a) 152/55 = 2.76, (b) 1.52e7/5.5e6 = 2.76, (c) |avg(30.4-45.6)/avg(11-16.5)| = 2.76</p>
<p>(as answered in comments)</p>
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https://biology.stackexchange.com/questions/30775/cationic-lipid-mediated-transfection-optimization-for-150mm-dishes
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Question: <p>I am interested to know if cysteine can form disulphide bridges in proteins within organelles. Typically cysteine will not form disulphide bonds in the reducing environment of the cytosol, but will in nonreducing environments such as the extracellular space.</p>
<p>From the <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/disulfide-bond" rel="nofollow noreferrer">Handbook of Biologically Active Peptides</a>:</p>
<blockquote>
<p>The ER is a vastly more common site than the cytosol (which is very rarely the site of disulfide bond formation) because the ER intralumenal environment is more oxidizing than that of the cytosol.</p>
</blockquote>
<p>This is fairly intuitive since the ER is where the disulphide bonds will be formed before translocation to the extracellular space. But what about other organelles?</p>
<p>Does this apply to all lumenal spaces as implied in the below picture, or is the ER a special case as a result of its necessity to fold proteins destined for the extracellular space?</p>
<p><a href="https://i.sstatic.net/Y9LVA.gif" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Y9LVA.gif" alt="enter image description here" /></a></p>
Answer:
|
https://biology.stackexchange.com/questions/101125/are-all-organelle-lumens-a-reducing-environment-like-the-cytosol-or-nonreducing
|
Question: <p>SO, as far as I understand, lysosomal hydrolyzing enzymes are first synthesized as proteins in rough ER and then they are budded off from the ER. The vesicles containing those proteins reach cis golgi and then they are properly folded to proper hydrolyzing enzyme but they are tagged with mannose-6-phosphate at this point, so they font have the ability to hydrolyze molecules just yet. In the mean time, some molecules can be engulfed by the cell and and these enter the cell as a membrane bound vesicle (endosome) containing proton pumps in its membrane which maintains an acidic environment inside the vesicle (which matures to late endosome). Vesicles containing hydrolyzing enzymes now move to those late endosomes and fuse with them. The low pH cleaves the mannose-6-phosphate tag from the enzymes and they become functional thus late endozome matures to lysosome. </p>
<p>The definition of primary lysosome says it is that lysosome that directly buds off from the golgi bodies and secondary lysosome is that lysosome which gets formed when primary lysosome binds with a vesicle and hydrolyse its content.</p>
<p>So are primary lysosme and those vesicles same?</p>
Answer:
|
https://biology.stackexchange.com/questions/59570/are-primary-lysosomes-same-as-vesicles-budded-off-from-golgi-bodies-containing-h
|
Question: <p>I would like to understand the meaning of the term <em>motif</em> as used in molecular biology.</p>
<p>In an article in <a href="https://www.nature.com/articles/nbt0406-423?proof=t#:%7E:text=Sequence%20motifs%20are%20short%2C%20recurring,and%20transcription%20factors%20(TF)." rel="nofollow noreferrer"><em>Nature Biotechnology</em></a>, Patrik D’haeseleer states:</p>
<blockquote>
<p>Sequence motifs are short, recurring patterns in DNA that are presumed to have a biological function. Often they indicate sequence-specific binding sites for proteins such as nucleases and transcription factors (TF).</p>
</blockquote>
<p><em>Does this mean that in the DNA sequence of a gene there are recurring patterns/subsequences of DNA which are presumed to have a biological function? If so, can such motifs be found simply by using a software that detects recurrent substring in a string?</em></p>
<p>In relation to the <strong>biological function</strong> of such motifs, I would like clarification of this extract from
<a href="https://pubmed.ncbi.nlm.nih.gov/16896524/" rel="nofollow noreferrer">a paper by Williams <em>et al.</em></a>:</p>
<blockquote>
<p><em>P. furiosus</em> ORF PF1193 displayed up to 12-fold increase in mRNA level at 20 min following irradiation (Table S1). PF1193 contains a ferritin-like di-iron <strong>motif</strong> found in ferritin- and Dps-like proteins and bacterioferritins and was found to belong to a new subclass of ferritin-like di-iron carboxylate superfamily (Ramsay et al. 2006; Tatur et al. 2005).</p>
</blockquote>
<p><em>By “motif”, do the authors mean that the gene PF1193 has a subsequence which is found many times in the DNA sequence of the gene that encodes ferritin- and Dps-like proteins, and that this may indicate that they (genes and the related proteins encoded) have similar characteristics/properties?</em></p>
<p>How does this relate to the sentence in the extract from the <em>Nature Biotechnology</em> paper?</p>
<blockquote>
<p>Often they indicate sequence-specific binding sites for proteins such as nucleases and transcription factors (TF).</p>
</blockquote>
Answer: <p><strong>Meaning of Motif in Molecular Biology</strong></p>
<p>In English the word, <em>motif</em> (borrowed from the French), has a variety of meanings in different areas. The one that is borrowed in molecular biology is that of <em>pattern</em> together with a hint, perhaps, of <em>emblem</em> or badge.</p>
<p>The word <em>pattern</em> indicates both repetition and a master mould from which copies are made. In molecular biology this indicates that this is not unique, it occurs repeatedly. The word <em>emblem</em> suggests a means of identifying the group to which something belongs. In molecular biology it is associated with a shared function for members of the group.</p>
<p><strong>Types of Motif in Molecular Biology</strong></p>
<p>Here the word <em>motif</em> is applied mainly to the three related macromolecules, DNA, RNA and protein. All of these are linear chains of restricted varieties of defined components (four nucleotides, 20 amino acids) arranged in defined ways which we refer to the sequence of the macromolecule. Within the overall sequence there can be sub-sequences, which, if they repeat represent patterns, and which may have functional significance. We refer to such patterns as:</p>
<ul>
<li>DNA sequence motifs</li>
<li>RNA sequence motifs</li>
<li>Protein (or amino acid)sequence motifs</li>
</ul>
<p>The first of these is what D’haeseleer is referring to. It should be noted that these sequence motifs can be absolute, or consist of <em>consensus sequences</em>, such as the one for the ROX 1-binding site in the article cited:<br>
<a href="https://i.sstatic.net/xoyXv.png" rel="noreferrer"><img src="https://i.sstatic.net/xoyXv.png" alt="Consensus sequence of ROX binding site" /></a>
<br>
However the nucleotides or amino acids are not the only components of macromolecules the arrangement of which can produce a pattern. Motifs in molecular biology can be composed of structural components. This is expressed in the <a href="https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_Basic_Cell_and_Molecular_Biology_(Bergtrom)/03%3A_Details_of_Protein_Structure/3.06%3A_Protein_Domains_Motifs_and_Folds_in_Protein_Structure" rel="noreferrer">following definition of protein motif</a>:</p>
<blockquote>
<p><em>Protein motifs</em> are small regions of protein <em>three-dimensional
structure or amino acid sequence</em> shared among different proteins. They
are recognizable regions of protein structure that may (or may not) be
defined by a unique chemical or biological function.</p>
</blockquote>
<p>And <a href="https://www.life.illinois.edu/mcb/150/private/faq/pdf/1390.pdf" rel="noreferrer">a similar definition</a> that considers only structural motifs in proteins adds: “An example… is a <a href="https://en.wikipedia.org/wiki/Helix-turn-helix" rel="noreferrer">helix-turn-helix motif.</a>” This is a motif in certain proteins that bind DNA.</p>
<p><strong>Identifying Sequence Motifs by Computer</strong></p>
<p>The limit of the approach suggested is that statistically one would expect any small sequence pattern to recur in DNA, and the problem is how to tell which recurring patterns have biological significance. It should be realized that is not the sequence motif alone that makes it functional, but the context in which it is found. Thus, a TATA box or other DNA motifs that act as binding sites for transcription factors were discovered (and are differentiated from other random or non-functional occurrences in the genome) by their proximity to the positions where transcription is initiated. Their function was confirmed experimentally, e.g. by binding RNA polymerase to the DNA. (This, I hope, answers the last query about the function of motifs as protein-binding sites.)</p>
<p>So, in general, no. Although I admit that I have personally use computational approaches to discover new small hydrogen-bonded protein motifs (a somewhat specialized area).</p>
<p><strong>The ferritin-like di-iron motif</strong></p>
<p>This is not a DNA sequence motif. It is not even a protein sequence motif, but a structural protein motif. The definition can be found on <a href="https://www.ebi.ac.uk/interpro/entry/InterPro/IPR009040/" rel="noreferrer">InterPro</a>:</p>
<blockquote>
<p>This entry represents a group of proteins, containing ferritin-like
domain, which is an about 145-residue domain made of a four-helix
bundle surrounding a non-heme, non-sulphur, oxo-bridged diiron site.
The diiron site is contained within a twisted, left-handed four-
helix-bundle constituted of two anti-parallel helix pairs connected
through a left-handed crossover connection.</p>
</blockquote>
<p>It is shown here with the helices coloured yellow and the iron atoms red spheres:</p>
<p><a href="https://i.sstatic.net/cLP1V.png" rel="noreferrer"><img src="https://i.sstatic.net/cLP1V.png" alt="The ferritin-like di-iron motif" /></a></p>
<p>[From <a href="https://www.nature.com/articles/nsb0696-539.pdf" rel="noreferrer">deMare <em>et al.</em> (1996)</a>]</p>
<p><strong>Footnotes</strong></p>
<ol>
<li><p>The helix–turn–helix is also referred to as a <em>domain</em>. The distinction between motif and domain is one of size (note the <em>small</em> in the definition of protein motif). However <a href="https://www.ebi.ac.uk/interpro/" rel="noreferrer">InterPro</a> does refer to the ferritin-like di-iron pattern as a motif, so this can be regarded as acceptable usage.</p>
</li>
<li><p>One might argue that as the sequence and structure of proteins are specified in the DNA, the motif should also be in the DNA. This is a fallacy. The information is there, but in a cryptic form. The redundancy of the genetic code means that protein sequences are far more conserved than the corresponding DNA sequences, and the three-dimensional structural patterns are not evident from inspecting amino acid sequences.</p>
</li>
</ol>
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https://biology.stackexchange.com/questions/101835/meaning-of-motif-in-molecular-biology
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Question: <p>I’ve been thinking about attributes that are unique to humans (not just far more developed in humans than other species) and aside from trivial things like chins, the only ones I can think of are art and language, but I’m not certain of either of these. Some songbirds sing for no apparent reason, which might suggest they have an intrinsic aesthetic sense.</p>
<p>As for language, what separates it from communication, I would argue, is grammar. It seems fairly clear that the other examples of what we would typically call communication in nature are not grammatical. But then I thought about DNA. People often speak metaphorically about DNA as a “programming language”. So, is that more than just a metaphor? That is, does DNA have clear lexical units, whose arrangement determines the relations between them? That is, is DNA, in whole or in part, grammatical?</p>
<p>There is a similar question <a href="https://biology.stackexchange.com/questions/29663/can-dna-rna-be-considered-as-natures-programming-language">here</a>, but the answers are too ambiguous in the use of the word “language” for my purposes. For example, English is undeniably a language. We could take all valid English sentences and order them as in a dictionary. This assigns each sentence a natural number. We could then assign the number the meaning of the corresponding English sentence. It would be fair to call this a “code”, but hard to call it a “language” in the strict sense: the digits of the numbers and their organisation would not really have meaning. In comparison, in English, the word “biology” has a specific reference, or placing an adjective before a noun has a specific meaning. I’m not talking about DNA “meaning” what it’s encoding in the sense that it knows what it’s doing, that is of course nonsense. Rather I’m using the word meaning in the sense that lexical units of DNA and ways to organise them would denote specific things, for example, specific ways to fold part of a protein, or bond amino acids. Just like how a computer doesn’t know what it’s code says, but still, in the psuedocode:</p>
<pre><code>while n>1:
if n even:
n = n/2
else:
n = 3n+1
print “Yes”
</code></pre>
<p>every term has a clear meaning, and their arrangement combines these meanings according to clear rules.</p>
<p>This may be an incredibly misguided question. I’m not a biologist.</p>
Answer:
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https://biology.stackexchange.com/questions/108797/is-dna-a-language
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Question: <p>I have mass spectrometry data (LC-MS/MS) from rat cortices under either drug or control treatments. The results were performed in triplicate (three pairs of rats, drug or control per pair). In addition to some of the bioinformatics analysis I am doing, I will have to validate some of the fold-changes from the mass spectrometry by Western blot in order to have my results published. </p>
<p>Some of the proteins I have chosen to to validate work very nicely by Western blot which captures the same trend as the mass spectrometry. However, other proteins that I have attempted to validate go in the exact opposite direction.</p>
<p>My questions: </p>
<ol>
<li>In general, how accurate is mass spectrometry compared to Western blotting?</li>
<li>Is it unusual to have a lot of variability between the results of the two methods? I am worried that I might have to try multiple proteins before I see the results replicated and this seems problematic to me. That leads me to the third question.</li>
<li>Which result should I trust?</li>
</ol>
<p>Other information - Some of the proteins I have chosen for validation come from lower in the list where not as many peptide fragments were detected. In some cases, fragments were detected in only 2 of the 3 biological replicates. I did not chose to validate proteins where the 2 or 3 replicates varied widely in normalized spectral count values so I would expect these readings to be accurate. Additionally, the mass spectrometry part of the experiment was performed by a reputable lab that has filtered erroneous data using FDR cutoffs. There is a high correlation overall between all three replicates for a particular treatment and low standard error between samples across the population so, statistically, I trust the mass spec results.</p>
<p>If you need more information please ask - I don't know what exactly will be useful to answer my questions. </p>
<p><strong>EDIT :</strong> </p>
<p><a href="http://www.mcponline.org/content/12/9/2381" rel="nofollow">This</a> article discusses the justification for using western blotting to validate mass spec results. It suggests using selected reaction monitoring assays as an alternative. Some of the points made by commenters in this thread are reiterated in this article.</p>
Answer: <p>It is a common practice to prove a result using an orthogonal technique. Like RNAseq followed by qRT-PCR etc.</p>
<p>Western blotting is not a robust technique and cross comparisons are difficult because of difference in the avidities/affinities of different antibodies. So comparisons can be made only with one protein-control pair in different conditions. </p>
<p>LC-MS is more sensitive and less biased(IMO). So the general trick is- don't do westerns for all proteins just report the ones that behave well (say that you "chose" these because they are important). I know this is a wrong practice and I should not be advocating it. For your own scientific validation try it with another MS technique; if you used ESI-Quadrupole/ion-Trap etc then try with MALDI-TOF or iTRAQ. Some luddites will continue to cling to westerns.</p>
<p>I think it is better to not test the proteins that have low peptide counts. See what is known as <a href="http://en.wikipedia.org/wiki/MA_plot" rel="nofollow">MA plot</a>. This is frequently used for microarrays. Here <code>M</code> denotes fold change and <code>A</code> denotes total expression in both samples. Don't pick proteins that are low in expression in both samples; they might not be meaningful. For example if you have <code>2</code> molecules of <code>X</code> and <code>100</code> molecules of <code>Y</code> in control condition and <code>5</code> molecules of <code>X</code> and <code>200</code> molecules of <code>Y</code> in test; then the fold change in <code>X</code> would seem important; however this change may not be relevant and can be a result of stochastic fluctuation/measurement error. If you have many samples you can see if something is stochastic or not but the limitation is the number of samples. </p>
<p><em>Note: I am not saying that 2→5 molecule increment should be meaningless. But to know if they have a meaning or not you would require more complex models; better avoid them at this moment.</em> </p>
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https://biology.stackexchange.com/questions/26028/mass-spectrometry-versus-western-blotting-for-validation
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Question: <p>In Life Ascending the author, Nick Lane, refers to an enzyme in his introduction:</p>
<p>'' <em>It concerns an enzyme (a protein that catalyses a chemical reaction) that is so central to life that it is found in all living organisms, from bacteria to man. This <strong>enzyme has been compared in two different species of bacteria, one living in superhot hydrothermal vents, the other in the frozen Antarctic. The gene sequences that encode these enzymes are different</strong>; they have diverged to the point that they are now quite distinct. We know that they did diverge from a common ancestor, for we see a spectrum of intermediates in bacteria living in more temperate conditions. But from the gene sequences alone there is little more we can say. They diverged, surely because their living conditions are so different, but this is abstract theoretical knowledge, dry and two-dimensional.
But now look at the molecular structure of these two enzymes, pierced by an intense beam of X-rays and deciphered through the wonderful advances in crystallography. <strong>The two structures are superimposable, so similar to each other that each fold and crevice, each niche and protrusion, is faithfully replicated in the other, in all three dimensions.</strong> An untutored eye could not distinguish between them.....The building blocks bind tightly to each other, through internal bonds that work like cement, retaining the structure despite the buffeting of energy from the boiling vents..... In the ice, the picture is reversed. Now the building blocks are flexible, allowing movement despite the frost...... <strong>Compare their activity at 6°C, and the frosty enzyme is twenty-nine times as fast; but try at 100°C, and it falls to pieces.</strong>
The picture that emerges is colourful and three-dimensional. The <strong>changes in gene sequence</strong> now have meaning: they <strong>preserve the structure of the enzyme and its function</strong>, despite the <strong>need to operate under totally different conditions</strong>.</em>''</p>
<p>Emphasis mine and .... indicates omitted text.</p>
<p>I would be very grateful if anyone could tell me what this enzyme is, and if possible point me to further information about it!</p>
<p>N. B. the enzyme is only mentioned in the introduction "... never found the opportunity for a mention in the book proper." There are no references for said introduction, only the chapters. </p>
<p>Thank you! </p>
Answer:
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https://biology.stackexchange.com/questions/79984/which-enzyme-is-nick-lane-referring-to
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Question: <p>Theoretically, mitochondria are said to be a separate organism that is concerned with its own life and its own processes. In fact, it even duplicates individually. I know a similar question is <a href="https://biology.stackexchange.com/questions/14347/the-origin-of-mitochondria">here</a> but I have something else apart from that to ask.</p>
<ol>
<li>Is the presence of their own DNA/RNA - like compound justify or prove that the mitochondria was once a separate living organism?</li>
<li>If the mitochondria is indeed a separate living organism, then how is it controlled by the nucleus? Or is it not? Logic would suggest that since mRNA exists in the mitochondria separate from DNA in the nucleus, they have their own control over how they behave.</li>
<li>Often, living organisms working together have to have some sort of payment system. For example, bacteria in the body provide Vitamin K and other breakdown vitamins, and in return the body gives it a suitable home to live in. Is there any such business occurring between the individual cell and the mitochondria? Does it keep some nutrients for itself? If not, what is its mode of nutrition? </li>
<li>In effect, cells did exist before mitochondria joined the party. Is there any way to at least have a feasible hypothesis on why and how the cells bonded with the mitochondria or how the cells even existed in the first place without the presence of and organnelle that can break down food and convert it into energy?</li>
<li>Why do mitochondria have their own membrane? And that too folded? Is that to increase the surface area available for the electron transport chain, or is it for simple compactness?</li>
<li>Why does the mRNA in our bodies always exactly match the mRNA of our mothers and only our <em>mothers</em>? Can any genetic mutation change this fact?</li>
<li>Is it possible, that a mitochondrion can somehow escape the cell and start performing its life otherwise?</li>
<li>mRNA would suggest that mitochondria make their own proteins / at least some basic amino acids. If there are any such proteins, are they utilized for the cell? Or are they used for cellular respiration? Or are they kept by the mitochondria for there own means?</li>
</ol>
<p>I will appreciate all answers!,
SmallDeveloper (a.k.a SmallScientist :) )</p>
<p>EDIT : Basically, I am asking 3 things:</p>
<ol>
<li>Were mitochondria really separate organisms once?</li>
<li>If so, how do they get their nutrition?</li>
<li>How did cells exist before mitochondria came along? </li>
</ol>
Answer: <ol>
<li>Yes. But it is incorrect to call mitochondria an organism now.</li>
<li>Most of their genes were lost and are now encoded in the nuclear genome</li>
<li>It gets most of its metabolites</li>
<li>It is not known. See the <a href="https://biology.stackexchange.com/questions/14347/the-origin-of-mitochondria">other</a> post for details.</li>
<li>Why membrane: I guess you know that. Why folded: you guessed right.</li>
<li>Only ovum donates mitochondria and other cytoplasmic factors. Sperm just provides the haploid genome.</li>
<li>No</li>
<li>Most of them are key respiratory enzymes</li>
</ol>
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https://biology.stackexchange.com/questions/20491/mitochondria-are-they-really-separate-organisms-that-once-merged-into-eukaryot
|
Question: <p>I saw this refutation online of Darwin's Random Evolution Theory and cannot see any holes with the logic. Can anyone crack this simple refutation?</p>
<blockquote>
<h1>Refutation of the Theory of Random Evolution</h1>
<p>As for the theory of evolution, which says that living things evolved progressively from
mud - first organism - bacteria - fish - animals - humans through tiny
random mutations which were advantageous and naturally selected;
there's a lot to say on this. All currently living life forms appears
to be highly related, sharing the same DNA system and cell structure.
This would suggest a common first ancestor as the theory suggests (or
better yet - one Designer), however, the most obvious flaw with the
theory is that the first organism must have had highly sophisticated
intelligent design. There is a minimum requirement for even the most
primitive possible life form, without which it could not possibly
survive.</p>
<p>Minimum Requirements for First Organism</p>
<ol>
<li><p>The first organism must have a system of producing and/or sourcing energy along with subsystems of distribution and management
of that energy which interact and work together, otherwise it cannot
power critical tasks such as reproduction.</p></li>
<li><p>It must have a system of reproduction which necessitates pre-existing subsystems of information storage (DNA), information
copying, and information reading/processing which interact with each
other and work together. This reproductive system is dependent on a
power source, so it must be coordinated with the power system. The
reproductive system must also copy/rebuild all critical infrastructure
such as the power system and the reproduction system along with the
"circuitry" and feedback mechanisms between them, otherwise the child
organism will be dead..</p></li>
<li><p>It must have a growth system, otherwise the organism will reduce itself every time it reproduces and vanish after a few generations.
This growth system necessitates subsystems of ingestion of materials
from the outside world, processing of those materials, distribution,
and absorption of those materials to the proper place, building the
right thing at the right place and in the right amount. It must also
have an expulsion system for waste materials.</p>
<p>The growth system must also be coordinated with the reproduction system. Otherwise, if the reproduction trigger happens faster than the
growth, it will reduce size faster than it grows in size and vanish
after a few generations. The growth system also requires connection to
the power infrastructure to perform its tasks.</p></li>
<li><p>All the "circuitry", signaling, and feedback infrastructure which allows the different systems and subsystems to coordinate
together and work together must be in place before the organism can
"come alive". The reproduction system won't work without coordination
with the growth and power systems. Likewise, the power system by
itself is useless without the growth and reproduction systems and
cannot survive. Only when all the "circuitry", etc. is in place and
the power is turned on is there hope for the hundreds of
interdependent tasks to start working together. Otherwise, it is like
turning on a computer which has no interconnections between the power
supply, CPU, memory, hard drive, video, operating system, etc -
nothing to write home about.</p></li>
<li><p>We assume it originated in water since gas is too unstable and solid is too static. If so, the organism must be contained by some
kind of membrane otherwise its precious contents will drift away in
the water due to natural diffusion or drifting of water due to
temperature variations in the water from sunlight, etc. or from heat
generated through its own power, or wind, moon, etc. If so, this makes
the assembly of such an organism more problematic, since it would need
to be closed shut before it can build itself in a stable way. Yet, to
build itself it would need to be open for a long time until all
systems are built and interconnected.</p></li>
</ol>
<p>From the above minimum requirements it is clear that the simplest
possible surviving organism is by no means simple. You would need
thousands of different proteins/lipids etc., in the right proportions,
all intricately folded and actively interacting with each other and
with sophisticated organelles. Contemplate this and you will see the
necessary complexity of this primitive organism is far more
sophisticated than anything modern technology has ever produced. Even
the most sophisticated Intel CPU is mere child's play compared with
the design of such an organism.</p>
</blockquote>
Answer: <p>I like @5th 's answer but I thought it might be worthwhile to clarify on some points and pull the logic out a bit more. </p>
<p>First there is some contesting the overall logic that it assumes all these <em>qualities of life are showing up at once</em>. If they had to, it probably is true that life could not evolve, but the general assumption is that there is a path to do so. Let me try to be convincing of this. </p>
<p>Dawkin's <a href="http://old.richarddawkins.net/articles/642589-first-life-the-search-for-the-first-replicator">theory of the replicator</a> is axiomatic and not entirely helpful at this point, but it does have a lot to say. Once you have self replicating systems, the rest of biology and evolution and selection does seem reasonable, even with the emergence of complicated structures like the <a href="http://musingsofscience.wordpress.com/2010/11/27/the-evolutionary-massif-and-the-eyes-pinnacle-part-1/">eye</a>, the <a href="http://www.detectingdesign.com/flagellum.html">flageller motor</a> and other seemingly unplausibly multi component systems seem to magically come together and do something new in the course of evolution. Dawkins wrote a book on this topic "<a href="http://en.wikipedia.org/wiki/Climbing_Mount_Improbable">Climbing Mount Improbable</a>" on this topic which tries to answer this question for several cases. </p>
<p>To outline why biological systems can and do seem to leap to new abilities, organs and unprecedented assemblies. In fact the do not, but there are so many possible trials (quintillions? ) over the trillions of years, that eventually some adaptive path to these features, which has usually been found when we go looking for it. As such there is no clear reason why new cases of this argument need to carry extra weight. The argument of statistically improbable combinations has been tried and remains unconvincing with respect to evolution several times. </p>
<p>After this, I think it would be fair to say that the origins of life, although coming clear still has many details to be resolved, but progress is being made. <a href="http://www.fossilmuseum.net/Paleobiology/Precambrian-Fossils.htm">Precambrian evidence of primitive life</a> was very different as seen in fossil records. We can see that at one time there were only bacteria and single celled organisms. There is evidence that at one time there appears to be only simple very large single celled organisms that lay in shallow water and soaked up the sunlight, growing and growing. </p>
<p>We can see that going back about 1.2-2 billion years, life shows a pretty clear progression where selection and adaptation creates complexity in living things and transitions life from exclusively chemotrophs (metabolizing Sulfur from geological processes) to anoxic (no oxygen environment) photosynthesis, and then oxygen breathing organisms. </p>
<p>In the precellular world, the RNA World hypothesis describes how all that life needed was one sort of molecule - RNA. That is chemically a very simple replicator. Evidence is pretty strong that RNA world was possible. Many chemists are interested in proving that RNA can be created more or less spontaneously from early earth chemical environments and recent experiments show that spontaneous <a href="http://www.nature.com/news/2009/090513/full/news.2009.471.html">soups containing RNA are a reasonable picture of earth at one point</a>. </p>
<p>All this being said its probably true that exactly what happened and how it happened will never be 100% known and the argument can be made that something unnatural happened at some point. But I hope you can also see that while the argument may still hold some water, the need for any magical interventions in the action of life is retreating to a point of origin - the beginning of time. Scientific inquiry is very much focused on filling the picture of the origins of life on earth though and given the success of finding probable paths through to the origins of life, the odds actually are in favor for making a statement of the physical mechanisms of life's origins. </p>
<p>Just a couple of side notes.. (1) biologists are not stuck on <a href="http://learn.genetics.utah.edu/content/epigenetics/inheritance/">everything being random</a>. There is lots of work on mechanisms evolved to <a href="http://learn.genetics.utah.edu/content/epigenetics/inheritance/">adapt in non-random way</a>s.<br>
(2) Darwin is not a sacred cow - just about any working biologist would love to show Darwin was wrong even in a little way. The same is true for Einstein, Newton and the rest. After a few years of trying though what one usually finds that its kinda difficult. Its not a matter of who makes the argument or what it means, but it has to be convincing and that <a href="http://www.gizmag.com/neutrinos-sub-light-speed/22876/">turns out to be difficult</a>. </p>
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https://biology.stackexchange.com/questions/6865/refutation-of-darwins-random-evolution-theory
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Question: <p>In what books can I find a detailed literature on the mechanism of function of different enzymes and proteins involved in DNA replication of <em>E. coli</em> ?</p>
Answer: <p>Check this online book on NCBI: <a href="https://www.ncbi.nlm.nih.gov/books/NBK21862/" rel="nofollow">An Introduction to Genetic Analysis. 7th edition</a></p>
<blockquote>
<p>Let’s examine each of these components and see how they fit together to produce our current picture of DNA synthesis in <em>E. coli</em>, the best-studied cellular replication system</p>
</blockquote>
<p>I used it a bunch while studying <em>E. coli</em> for my master degree, lots of really usefull information.</p>
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https://biology.stackexchange.com/questions/52570/books-dna-replication
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Question: <p>When the DNA replicates, it first attaches RNA since the DNA polymerase can't attach DNA in the 3' end. Why the replication is happened this way?
If the DNA polymerase can attach DNA from the 3', the replication process will be much simpler.
Is there a special reason for this?</p>
Answer:
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https://biology.stackexchange.com/questions/65583/dna-replication-why-complex
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Question: <p>Why does nature rely on RNA primer for the start of DNA Replication? Why not simply use DNA primer and make life simple !</p>
Answer: <p>Biochemically none of the DNA-dependent DNA polymerases involved in DNA replication have the ability to begin elongation without a 5' to 3' primer. </p>
<p>The only DNA Polymerase that can catalyze elongation without a 5' to 3' primer is Reverse Transcriptase, however Reverse Transcriptase is an RNA-dependent DNA polymerase, and that accounts for the difference.</p>
<p>And if your next question is, but why, the answer is that that is how the systems evolved. Also DNA Polymerases have a much higher rate of fidelity due to error checking than RNA Polymerases do, and as the RNA primers get removed anyway and replaced with DNA, an error here or there in the primer will not matter. </p>
<p>The RNA-DNA duplex is also less stable than the DNA to DNA duplex, so it makes it easier for FEN1 to remove an RNA Primer than it would a DNA primer, assuming that the mechanism of primer removal and replacement after the complementary leading and lagging strands have been synthesized remained in place.</p>
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https://biology.stackexchange.com/questions/39007/dna-replication-and-primer
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Question: <p>This will sound as a super stupid question, but I just read in the <em>Molecular biology of the gene</em> book (7th edition, Watson, Baker, Bell and al.) that <strong>one mistake occurs in 10 million nucleotides added during the replication of DNA</strong>.</p>
<p>However, I read in the Campbell's biology book (11th edition) that it is <strong>one mistake in 10 billion nucleotides added</strong>.</p>
<p>I tried to google it and kept finding these two numbers... probably because people use the same books as I do as sources. So, my question is the following: <strong>which book is right about DNA replication precision?</strong></p>
<p>Thanks for your help and sorry if my question isn't very interesting.</p>
Answer:
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https://biology.stackexchange.com/questions/77039/dna-replication-precision
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Question: <p>Is DNA replication required for Protein Synthesis or can proteins be synthesized without DNA being replicated?</p>
Answer: <p>DNA replication and protein synthesis are very very different processes. The key thing to remember about protein synthesis is that DNA is not directly used; RNA is. When DNA is replicated, it is for reproduction. When it is transcribed, it is copied, with certain changes, into RNA instead. RNA is then "read" by protein synthesis machinery to produce the proteins. </p>
<p>To directly answer your question, no, replication is not required. If you inject some RNA which codes for a protein into a cell, it will be translated into a protein. There are many viruses which exclusively use RNA for their genetic makeup. The RNA is replicated at some point by the host cell's machinery but the RNA can be directly translated into a protein by the host cell as soon as the RNA enters the cell.</p>
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https://biology.stackexchange.com/questions/77749/dna-replication-and-protein-synthesis
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Question: <p>Just out of curiosity (I am completely strange to biology), as I have been unable to find this info on the internet:
How long does the whole DNA replication process take? (say, the replication of a whole chromosome)
Approximately, how long does the DNA spend in a single helix structure during replication? </p>
<p>I am curious about the time scales of these processes, from the moment the polymerase starts cutting the double helix till the moment there are two double helices perfectly formed (one of them a copy of the other). How long can this take? miliseconds? seconds?</p>
Answer: <p>A mammalian cell takes about <strong>8 hours</strong> to replicate all of its DNA in its <em>S phase</em>; a yeast cell would take about <strong>40 minutes</strong>.</p>
<p>Some other information that you seem to not have quite the right information about:</p>
<ol>
<li><p>The DNA polymerase does not unwind/split the DNA—that is the job of <em>DNA helicase</em>. The DNA polymerase cannot bind to the single-stranded DNA until the helicase unwinds the double-stranded DNA ("melts"), or the dsDNA is artificially melted in a test tube via high temperature.</p>
</li>
<li><p>The dsDNA is not fully separated before it is copied. There are tens of thousands of sites—<em>origins of replication</em>—across the genome that unwind and begin replicating every time the cell replicates its genome.</p>
</li>
</ol>
<blockquote>
<p>"...DNA replication in most eukaryotic cells occurs only during a specific part of the cell-division cycle, called the <em>DNA synthesis phase</em> or <em>S phase</em>. In a mammalian cell, the S phase typically lasts for about <strong>8 hours</strong>; in simpler eukaryotic cells such as yeasts, the S phase can be as short as <strong>40 minutes</strong>."</p>
<p>"An average-size human chromosome contains a single linear DNA molecule of about 150 million nucleotide pairs. It would take (0.02 seconds/nucleotide) x (150e6 nucleotides) = 3e6 seconds (about 35 days) to replicate such a DNA molecule from end to end <em>with a single replication fork</em>."</p>
<p>"...Approximately 30,000–50,000 origins of replication are used each time a human cell divides."</p>
</blockquote>
<p>–Molecular Biology of the Cell, 6e, Alberts, et al.</p>
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https://biology.stackexchange.com/questions/81107/typical-dna-replication-times
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Question: <p>I just wanted to understand the basic steps behind the replication of the lagging strand of DNA:</p>
<ul>
<li>Have helicase unwind it first</li>
<li>DNA Primase lays down RNA primers in fragments, called Okazaki fragments</li>
<li>DNA polymerase III goes through and corrects all the mistakes (essentially replace the Uracil with Thymine)</li>
<li>DNA polymerase I goes through and removes RNA primer</li>
<li>DNA ligase hooks up each of the 5' to 3' end fragments</li>
</ul>
<p>Is this the correct order of steps? If so, does the leading strand need any RNA primer or does the DNA polymerase just start and go to the end without any help?
Thanks</p>
Answer: <p>I think that you have a couple of points wrong. Since your question is asked using bacterial terminology, I'll stick to that.</p>
<p>The leading strand, the one that is initiated at the origin of replication, is synthesised by pol III which is a highly processive polymerase, i.e. it keeps on going for long periods, making very long products. In principle a single pol III molecule could produce the entire leading strand copy of the template.</p>
<p>The lagging strand is, as you say, initiated at multiple RNA primers. The enzyme that extends these primers is pol I. As polI extends a primer it creates an Okazaki fragment (i.e. this is not an alternative name for the primer itself). Eventually the pol I will encounter the 5' end of another RNA primer. At this point the 5'>3' exonuclease activity of the pol I comes into play and removes the primer, replacing it with DNA. The pol I will probably also degrade some of the DNA that has been added to that primer by another pol I, resynthesising it as it goes. This is so-called "nick tranlation" since as the pol I moves along the template it moves a nick in the new strand as it goes. pol I is not a very processive enzyme however and will fall off, leaving the nick to be sealed by DNA ligase.</p>
<p>The leading strand does require a primer, and in most cases this is an RNA laid down by the initiation complex at the origin of replication. In some cases, a protein provides an -OH group for DNA polymerase to initiate at. </p>
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https://biology.stackexchange.com/questions/9111/dna-replication
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Question: <blockquote>
<p>The E.coli DnaB helicase is essential for replication initiation from the chromosomal origin of replication ( oriC ) and is present in vivo as a protein complex with six monomers of the DnaC ATPase protein and six ATP molecules (Wickner and Hurwitz, 1975; Lanka and Schuster, 1983). DNA replication initiation at oriC begins with binding of multiple molecules of the bacterial DnaA initiator protein to a 9 bp repeats (DnaA boxes). This binding promotes destabilization of nearby AT-rich sequences, resulting in unwinding of the DNA double helix and the formation of an <strong>open complex</strong>.</p>
</blockquote>
<p>I looked up in DNA Replication by Arthur Kornberg, Tania A. Baker (the authors who probably coined this term), Google and Google.Scholar but didn't stumble upon any definition. </p>
<p>What is it actually?</p>
Answer: <p>The sentence itself is actually the definition of <em>open complex</em>: It is the structure that is created once the DNA double helix is unwinded due to the DnaA proteins.</p>
<blockquote>
<p>This binding promotes destabilization of nearby AT-rich sequences,
resulting in unwinding of the DNA double helix and the formation of an
open complex.</p>
</blockquote>
<p>I have also found a paper<sup>a</sup> co-authored by Arthur Kornberg himself where the authors self-define "open complex" as well:</p>
<blockquote>
<p>Three tandem repeats of a 13-mer in the AT-rich region are essential
to the unique replication origin of E. coli and of remotely related
Enterobacteriaceae. These iterated sequences are identified by
deletion analysis and sensitivities to endonucleases as the site for
initial duplex opening by the initiator dnaA protein. This “open
complex” requires ATP and 38% for optimum formation and stability.</p>
</blockquote>
<p>There are several papers<sup>b,c</sup> that use "open complex" in the same way.</p>
<p><strong>References:</strong></p>
<p><sup>a</sup> David Bramhill, Arthur Kornberg, Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome, Cell, Volume 52, Issue 5, 1988, Pages 743-755, ISSN 0092-8674, <a href="http://dx.doi.org/10.1016/0092-8674(88)90412-6" rel="nofollow noreferrer">http://dx.doi.org/10.1016/0092-8674(88)90412-6</a>.
(<a href="http://www.sciencedirect.com/science/article/pii/0092867488904126" rel="nofollow noreferrer">http://www.sciencedirect.com/science/article/pii/0092867488904126</a>)</p>
<p><sup>b</sup> Ozaki, Shogo, et al. "A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin." Journal of Biological Chemistry 283.13 (2008): 8351-8362. (<a href="http://www.jbc.org/content/283/13/8351.full" rel="nofollow noreferrer">http://www.jbc.org/content/283/13/8351.full</a>)</p>
<p><sup>c</sup> Mukhopadhyay, Gauranga, et al. "Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication." The EMBO journal 12.12 (1993): 4547. (<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC413885/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC413885/</a>)</p>
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https://biology.stackexchange.com/questions/54386/what-is-open-complex-in-e-coli-dna-replication
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Question: <p>I’m currently learning about DNA replication in both prokaryotic and eukaryotic cells. And my lecturer has mentioned that replication is a once in a lifetime activity. And I’m not sure what this is implying because I’ve searched up that DNA replication occurs during cell division (cell cycles), which occur repetitively as organisms develop.</p>
Answer: <p>This sounds like a difference in perspective of when exactly cells "die". If you consider that, in a cell division, a mother cell "gives birth" to two daughter cells, you could argue that the mother cell has "died". It makes sense to think about cell division in this way because it puts both daughter cells in the same level, without one being "special" due to it being the mother cell itself while the other is considered the "new cell".</p>
<p>If you face cell division like this, any cell (eukaryote or procaryote) will only really duplicate their DNA once: they duplicate their DNA -> they split into daughter cells, in which they "die" -> (eventually) each daughter cell duplicate their DNA -> and so on.</p>
<p>Like I said this is just a particular POV we can adpot when we try to understand and describe cell cycle and cell division (although it is a common one for researchers in the field).</p>
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https://biology.stackexchange.com/questions/81875/dna-replication-how-many-times-and-when-does-it-occur
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Question: <p>I am a bit confused.
During Meiosis, DNA is replicated to form a cell with half the DNA and likely to have variations.
But since the replication process of meiosis and mitosis are the same, why do DNA in different cells of an individual organism not have variations? Or do they have variations that are just not expressed?</p>
Answer: <p>Think of Meiosis as a means of producing half the genetic material you (and your partner) need which, once combined, produces offspring that inherits genetic material from the both of you. Think of Mitosis as a means of producing a full, exact (as exact as possible anyway) copy of chromosomes during cell division =)</p>
<p><strong>Meiosis</strong> - results in a cell with half the genetic material (haploid), which is later fused with a similar haploid cell from the opposite sex (gametes) to produce a single cell with a full set of chromosomes (diploid cell). I.e. a sperm and an egg (each haploid) which fuse to produce a zygote (diploid). Important to note that during Meiosis, there is cross over between chromosomes, so that you end up with genetically diverse gametes (i.e. each haploid cell is different to one another - part of the reason your kids don't all look exactly the same).</p>
<p><strong>Mitosis</strong> - results in a cell with a full genetic copy of the cell it was replicated from. That zygote will replicate using mitosis again and again until the organism dies. During Mitosis the cell tries to preserve as much of it's integrity as possible, so there isn't cross over. But the process isn't perfect in humans - each division results in shorter telomeres, and a cells also accumulate mistakes that result in "bad code" being passed on - UV damage for example.</p>
<p>With regards to expression, there is the concept of <a href="https://en.wikipedia.org/wiki/Epigenetics" rel="nofollow noreferrer">epigenetics</a> - you may inherit certain genes, which (through various mechanisms) will be "locked" and not expressed in yourself, but may be expressed in your children. You might notice that kids have characteristics of their grandparents which aren't present in their parents.</p>
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https://biology.stackexchange.com/questions/70508/dna-replication-during-mitosis
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Question: <p>My Campbell's Biology textbook contains the following diagram related to the semi-conservative model of DNA replication proposed by Watson and Crick. I have highlighted where my confusion arises in red:</p>
<p><a href="https://i.sstatic.net/I2njo.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/I2njo.jpg" alt="enter image description here"></a></p>
<p>So, I understand what goes on in the first replication--that's pretty much straightforward. However, what I don't understand is why in the second replication, the light blue strand isn't paired with a dark blue strand. After all, aren't the light blue strand and dark blue strand complementary, per the results of the first replication? </p>
<p>After the light blue and dark blue DNA strands separate to serve as templates in Replication #2, why don't we see two dark+light double-helices?</p>
Answer: <p>I believe the reason you are having trouble understanding the concept is due to a poor usage of colors in the diagram. Don't focus on the colors, but on the concept. It's the same for both replication events. Each strand of a double helix is used as a template to make a new complimentary strand, giving rise to two new DNA helices from the original. In each new double helix, one strand should match that of the double helix it came from before, as it was the template and is the <em>same</em> strand. While the other strand, the complimentary strand, has been newly synthesized to match the complimentary strand. In this diagram, every newly synthesized strand is shown in light blue.</p>
<p>Start by looking at the first double-helix of DNA (navy blue). During replication, the DNA is unwound and each navy strand is used as a template to create the newly synthesized (complimentary) strand, which is shown in light blue. In this first all navy double helix, you have two navy strands that are <em>each</em> used as a template strand and new complimentary strands (light blue) are synthesized to match . This produces the second two helices (navy and light blue). They each have one strand from the original helix (navy), and one new strand (light blue). </p>
<p>The process is exactly the same for the second replication event, only, a new color was not introduced to show a newly synthesized strand, so one cannot differentiate between the original (template) strand and the new complimentary strand. Both are pictured as light blue. </p>
<p>For the final replication event, only focus on the top navy and light blue helix first. Just as before, the two strands are separated from each other and are <em>both</em> used as templates to create a new strand. The navy blue strand is used as a template for a new light blue strand to be transcribed. This produces the top-most helix, where the original strand is shown as navy and the newly transcribed (complimentary) strand is light blue. </p>
<p>Just as the navy strand was a template, the light blue strand from the bicolored helix is used as a template strand as well to create the second all light blue helix. This light blue template strand is used to synthesize a new light blue strand, creating the all light blue helix. One of the strands is the original light blue one from the navy and light blue helix, while the other strand is the newly synthesized one. </p>
<p>It might've been easier if they had used a different color for the final round of replication, like orange. Then to show the newly synthesized strands in the final round, the 1st and 4th double helix would consist of a navy (original) strand and an orange (newly synthesized) strand. The second and third double helices would then have each had a light blue (original) strand and an orange (newly synthesized) strand. This would have made it easier to understand where each strand originally came from. </p>
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https://biology.stackexchange.com/questions/42459/the-semi-conservative-model-of-dna-replication-question
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Question: <p><strong>Background research</strong></p>
<p>I am aware that DNA polymerase <a href="https://en.wikipedia.org/wiki/DNA_polymerase" rel="nofollow noreferrer">works in pairs</a>, at least. This is to process both opposite stands of a given chromosome. The 3'-to-5' "leading strand", and 5'-to-3' "lagging strand" simultaneously. This two-DNA-polymerase process is beautifully shown in this <a href="https://www.youtube.com/watch?v=WFCvkkDSfIU&t=03m35s" rel="nofollow noreferrer">TED talk by Drew Berry</a>.</p>
<p>I have not checked any non-online sources but simple Google queries failed to answer my questions below. </p>
<p><strong>Question</strong></p>
<p>Within a single Eukaryotic cell, is the process of DNA-replication carried out by just a single pair of DNA-polymerases? Or do multiple pairs of DNA-polymerases work in parallel? </p>
Answer: <p>In the (<em>beautifully</em> rendered) video you linked to, the green molecules are DNA polymerases. So you can already see that there are more than two DNA polymerases at work!</p>
<p>At each replication fork, there is generally one DNA polymerase working on the leading strand, but on the lagging strand, multiple DNA polymerases may be working at the same time (as depicted in the video). Note that different types of polymerase are thought to be primarily responsible for the leading (<a href="https://en.wikipedia.org/wiki/DNA_polymerase_epsilon" rel="nofollow">Pol ε</a>) and lagging (<a href="https://en.wikipedia.org/wiki/DNA_polymerase_delta" rel="nofollow">Pol δ</a>) strands in eukaryotes (but not prokaryotes, which use <a href="https://en.wikipedia.org/wiki/DNA_polymerase_III_holoenzyme" rel="nofollow">Pol III</a> for both).</p>
<p>Next, note that at every <a href="https://en.wikipedia.org/wiki/Origin_of_replication" rel="nofollow">origin of replication</a>, there are two replication forks working simultaneously, one in each direction. So that's double the number of polymerase molecules.</p>
<p>On top of that, a single eukaryotic cell has multiple chromosomes. Each one must have at least one origin of replication of its own, which can operate in parallel. In reality, each eukaryotic chromosome has up to thousands of origins of replication (prokaryotic chromosomes generally only have one). Human cells have on the order of 100,000 origins of replication. So depending on how many origins are active simultaneously, there are likely many thousands of polymerase molecules acting at once to replicate the DNA of a single eukaryotic cell.</p>
<p>You could probably do a rough estimate of the number of polymerase molecules active simultaneously by taking the average duration of S phase in your cell of interest and dividing by the genome size and the speed of polymerase.</p>
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https://biology.stackexchange.com/questions/52811/dna-replication-how-many-dna-polymerase-molecules-work-in-parallel
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Question: <p>In diploid organisms like humans, germ cells typically undergo DNA replication before meiosis. This is followed by two rounds of cell division (meiosis I and II), ultimately producing four haploid gametes.</p>
<p>I am curious about why DNA replication before meiosis is necessary.</p>
<p>Is it possible for a diploid organism to have germ cells that skip DNA replication and undergo only <strong>one</strong> division instead of two, directly producing <strong>two</strong> gametes instead of four? If not, what biological constraints make this impossible?</p>
Answer: <p>As a matter of fact, yes. Though extremely rare, there are a few organisms that undergo single-division meiosis. As far as I can tell, this has only been identified in "some flagellates (<a href="https://en.wikipedia.org/wiki/Parabasalid" rel="noreferrer">parabasalids</a> and <a href="https://en.wikipedia.org/wiki/Oxymonad" rel="noreferrer">oxymonads</a>) from the gut of the wood-feeding cockroach <a href="https://en.wikipedia.org/wiki/Cryptocercus" rel="noreferrer">Cryptocercus</a>."<a href="https://en.wikipedia.org/wiki/Meiosis#Flagellates" rel="noreferrer"><sup>Wikipedia</sup></a></p>
<p>The reference in the Wikipedia article points to Raikov (<a href="https://www.sciencedirect.com/science/article/pii/S0932473911803494?via%3Dihub" rel="noreferrer">1995</a>), which is a review of meiosis more broadly, and Raikov cites Cleveland (<a href="https://doi.org/10.1111/j.1550-7408.1956.tb02452.x" rel="noreferrer">1956</a>). Raikov proposes that single-division meiosis in these groups is of a secondary origin (e.g., they have ancestors that did 2-division meiosis), and must be a "blind branch of evolution since one-divisional meiosis of the same type occurs nowhere else and because intestinal parasites of insects are surely a specialized group."</p>
<p>There has been some discussion about why 2-division meiosis is so prevalent. Archetti (<a href="https://doi.org/10.1111/j.1420-9101.2004.00726.x" rel="noreferrer">2004</a>*) states in his abstract, "one‐step meiosis is very rare in nature, and may not even exist at all. I suggest that this is because one‐step meiosis, in contrast to two‐step meiosis, can be easily invaded and replaced by asexual reproduction."</p>
<hr />
<p>*Unfortunately, this article is paywalled.</p>
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https://biology.stackexchange.com/questions/116200/do-any-diploid-organisms-skip-dna-replication-and-undergo-only-one-division
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Question: <p>Assuming that all environmental conditions on Earth remain the same in distant future, the tendency of nature to increase entropy would cause the chemistry and the mechanism of DNA replication to create more and more "errors".</p>
<p>Could these errors accumulate over time for all life, resulting in failure of replication by means known today, bringing life as we know it to extinction?</p>
<p>In other words, will life eventually fail to continue reproducing due to the inability to maintain its inherent state of non-equilibrium?</p>
Answer: <p>The principle that entropy (disorder) must increase is true for a closed system. An organism, however, is not a closed system; it obtains energy from outside itself and with that energy can maintain order. This is referred to as <em>negative entropy</em>; see the <a href="https://en.wikipedia.org/wiki/Entropy_and_life#Negative_entropy" rel="nofollow noreferrer">Wikipedia article on Entropy and Life</a>.</p>
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https://biology.stackexchange.com/questions/95446/could-dna-replication-fail-in-the-far-future
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Question: <p>E.coli divides in 20 minutes and its DNA replicates in 38 minutes .Kindly explain.</p>
Answer:
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https://biology.stackexchange.com/questions/39499/e-coli-division-and-its-dna-replication
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Question: <p>Could someone help me understand this equation please? I found it in a paper which said that it was DNA replication, but why?</p>
<p>$\ce{dNTP + dNMP_{n} -> dNMP_{n +1} + PPi}$</p>
<p>I found that <strong>dNTP</strong> means <em>deoxy nucleotide triphosphate</em> and <strong>dNTP</strong> means <em>deoxy nucleotide monophosphate</em>. Deoxy nucleotide monophosphate is a monomer in DNA, but dNTP isn't.</p>
<p>I also would like to know if there is an equation like this in other processes in the central dogma (such as transcription and translation)?</p>
Answer: <p>The notation you are referring to is <em>a</em> way to express the elongation of a nucleotide strand (Fig. 1).</p>
<pre>dNTP + dNMP<sub>(n)</sub> → dNMP<sub>(n+1)</sub> + PP<sub>i</sub> </pre>
<p>means</p>
<p><strong>Existing strand + deoxynucleotidetriphosphate → elongated-strand + pyrophospate.</strong></p>
<p><img src="https://i.sstatic.net/X7K0M.jpg" alt="elongation"> </p>
<p><sup>Fig. 1. Elongation of DNA. Source: <a href="http://bio3400.nicerweb.com/Locked/media/ch11/chain_elongation.html" rel="nofollow noreferrer">Concepts in Genetics</a>.</sup></p>
<hr>
<p>This reaction holds for DNA replication as well as transcription. </p>
<p>Translation is all about protein synthesis from amino acid precursors. Each subsequent amino acid is coupled to the growing peptide by a peptide bond between the carboxyl of the growing peptide and the amino group of the new amino acid. A water molecule is eliminated during the reaction (Fig. 2).</p>
<p><img src="https://i.sstatic.net/008sP.png" alt="peptide synthesis"><br>
<sup>Fig. 2. Peptide bond formation. Source: <a href="https://en.wikibooks.org/wiki/An_Introduction_to_Molecular_Biology/Function_and_structure_of_Proteins" rel="nofollow noreferrer">An Introduction to Molecular Biology</a></sup></p>
<p>Hence translation can be written analogously to replication as:</p>
<pre>
amino acid + peptide<sub>(n)</sub> → peptide<sub>(n+1)</sub> </pre>
<p>However, also in this polymerization reaction energy is needed and amino acids are <a href="http://www.nobelprize.org/educational/medicine/dna/a/translation/trna_aminoact.html" rel="nofollow noreferrer">activated</a> while they are bound to the messenger RNA (mRNA) through the action of ATP. Moreover, during translation the <a href="http://bscb.org/learning-resources/softcell-e-learning/ribosome/" rel="nofollow noreferrer">ribosome (i.e., the protein synthesizing machinery)</a> uses another source of energy to move across the RNA template, namely GTP. Hence, the <em>net</em> reaction can be written as:</p>
<pre>
amino acid + peptide<sub>(n)</sub> + ATP + GTP → peptide<sub>(n+1)</sub> + AMP + GDP + PP<sub>i</sub> + P<sub>i</sub> </pre>
<p>Note that this notation in itself is <em>not</em> a dogma as other ways are in use to denote DNA and RNA synthesis. According to <a href="http://www.merriam-webster.com/dictionary/dogma" rel="nofollow noreferrer">Merriam-Webster</a> a dogma is:</p>
<blockquote>
<p>A belief or set of beliefs that is accepted by the members of a group without being questioned or doubted.</p>
</blockquote>
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https://biology.stackexchange.com/questions/35621/what-does-this-equation-about-dna-replication-mean
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Question: <p>So I am taking a course in DNA replication and repair. And we are talking about catenanes forming when DNA replicates (two circles of dsDNA interlinked) How is this possible?</p>
Answer: <p>The first DNA circle is double-stranded. If you could melt the double-helix completely you would not be able to pull the two stands apart without breaking the sugar-phosphate backbone of at least <em>one</em> of the two strands. This is a topological problem, the two strands are linked to each other.</p>
<p>Now consider DNA replication. In the simplest example there is a single origin of replication and there will be two replication forks proceeding around the circular genome in opposite directions. This is exactly what happens during DNA replication in the bacterium <em>E. coli</em>. The DNA double helix of the parental molecule "melts", the RNA primers are synthesized by primase, and the DNA polymerase complexes initiate synthesis. Behind each replication fork there are now two hybrid DNA strands, each with a parental strand, and a new daughter strand.</p>
<p>Thus far no sugar-phosphate bonds have been cleaved. When replication is done each daughter ds DNA helix will have a single-stranded nick at the origin, and another one at the site where replication terminated, but those will be quickly repaired by DNA polymerase I and DNA ligase. Even if those two repair enzymes were inhibited, and you could somehow melt the DNA strands of both the daughter chromosomes, you would only be able to pull out the new DNA molecules (with the nicks), the two original parental strands are <strong>still</strong> topologically linked.</p>
<p>To resolve two concatenated DNA circles you need to make a double-stranded break in at least one of the ds circles. This enzymatic activity is provided by a class of enzymes named Type II DNA topoisomerases.</p>
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https://biology.stackexchange.com/questions/36811/how-do-catenanes-form-when-dna-replicates
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Question: <p>Let's say we have two <strong>DNA molecules of equal length</strong>, one belonging to a prokaryote and the other to an eukaryote. It's known that replication of the eukaryotic DNA is faster in this case. One clear reason for this is that linear DNA has multiple origins of replication whereas circular DNA only has one.</p>
<p>Now back to the real question: Does it matter for rate of replication whether the DNA is circular or linear? <em>Does it contribute to eukaryotic DNA replication being faster <strong>in our specific case</strong>?</em></p>
<p>One thing I found in an old revision of this <a href="https://en.wikipedia.org/wiki/Linear_chromosome" rel="nofollow noreferrer">Wikipedia article</a> is as such:</p>
<blockquote>
<p>One reason that many organisms have evolved to having linear chromosomes is due to the size of their genome. Linear chromosomes make it easier for transcription and replication of large genomes. <strong>If an organism had a very large genome arranged in a circular chromosome, it would have the potential problems when unwinding due to torsional strain.</strong></p>
</blockquote>
<p>What I make out from this is that the rate of replication <em>isn't</em> directly affected. It's more closely related to avoiding other issues that arise from eukaryotic DNA molecules being <em>typically</em> longer than prokaryotic ones. But in our case where we declare both DNA to be of equal length, I'm assuming circular vs linear has no bearing on the rate of DNA replication.</p>
<p>So am I correct on my assumptions?</p>
<h1><strong>Edit in response to the answer below:</strong></h1>
<blockquote>
<p>It's known that replication of the eukaryotic DNA is faster in this
case (where DNA molecules are of equal length) <strong>because eukaryotes
have linear chromosomes whereas prokaryotes have circular ones</strong></p>
</blockquote>
<p>If it makes sense to form a sentence like this, presenting linearity as the cause, then it's enough to satisfy my definition of <em>directly affected</em> in this case. For example, it's easy to make this claim if you present the number of origins of replication as the cause. I looked at the textbook you mentioned and the quantity of origins of replication is in fact mentioned this way.</p>
<h3>Conclusion:</h3>
<p>I went ahead and did a bit more reading in that textbook. What
I've understood is: An eukaryotic linear chromosome has multiple
replication origins rather than a single one in order to compensate
for its much larger size. So while the shape might be a factor
(possibly, not certainly), the primary and "direct"
reason is the difference in size, not the shape.</p>
Answer: <p>I am not sure if I well understand what you mean by <strong>directly affected</strong>.</p>
<p>I will list some possibilities below (For reference you can see any genetics textbook, I use, Genetics: A conceptual approach by Pierce, but I guess any textbook would do).</p>
<ol>
<li><p>Porkaryotic polymerases (<em>usually</em> processing circular DNA) have a higher nucleotdie per second speed than eukaryotic polymerases (processing linear DNA). Does this mean that shape <strong>directly affects</strong> speed? In this case I would say no. It's just a difference between prokaryotic and eukariotic polymerases. Some prokaryotes have linear chromosomes and their polymerase will still be faster than eukaryotic polymerases.</p>
</li>
<li><p>One prokaryotic chromosome has only one origin of replication, while one eukaryotic chromosome has several of them, so that eukaryotes parallelize even in a single chromosome. Does this mean that shape <strong>directly affects</strong> speed? In this case, I would say yes. Due to steric effects, the shape of the chromosome has to do with the ability of having several origins of replications, even if the lengths are the same. <strong>Edit</strong>: After <a href="https://biology.stackexchange.com/users/60767/ved">Ved</a>'s comment, I realized that this is also not necessarily true. Not all circular chromosomes have one single origin of replication; archaea have circular chromosomes with <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3783049/" rel="nofollow noreferrer">more than one origin of replication</a></p>
</li>
<li><p>Linear chromosomes are easier to unwind. Does this mean that shape <strong>directly affects</strong> speed? I would say, yes for large chromosome and no for small chromosomes. <strong>Edit</strong>: After <a href="https://biology.stackexchange.com/users/22057/david">David</a>'s comment, I realized that I am not really sure that linear chromosomes are easier to unwind. A Wikipedia <a href="https://en.wikipedia.org/wiki/DNA_unwinding_element" rel="nofollow noreferrer">entry</a> states this, but I gave a look at the <a href="https://www.annualreviews.org/doi/pdf/10.1146/annurev.bi.62.070193.000333?casa_token=1xzBx_8MY8wAAAAA:kMm-y_nYz9t2O_Rptb8nWNZOSjPWWz5Ctyhiq6ILMU6b2WRApzQ_OK_KNIec33-qc9qHQ_698eg" rel="nofollow noreferrer">paper they referenced</a> and found no real statement about this.</p>
</li>
</ol>
<p>Importantly, if the DNA molecules you compare are one prokaryotic and one eukaryotic and they are very short, I would bet my 5 bucks on prokaryotic (circular) being faster, because in absence of all the parallelization the eukaryotic DNA polymerases are <strong>slower</strong> than prokaryotic ones.</p>
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https://biology.stackexchange.com/questions/94552/does-dna-being-circular-or-linear-directly-affect-the-speed-of-dna-replication
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Question: <p>During DNA replication, RNA primase puts an RNA primer in the lagging strand. What is the function of this RNA primer? Why can't the enzymes put DNA fragments directly?</p>
Answer: <p>DNA polymerases need a primer oligonucleotide (RNA or DNA) - their substrates are an existing 3'-OH group and a dNTP. The primase however is a typical RNA polymerase, capable of initiating polynucleotide synthesis <em>de novo</em> by positioning a complementary ribonucleoside 5'-triphosphate opposite its complementary DNA base. The primase makes an RNA primer that the DNA polymerase can then use for chain extension. The RNA primer is ultimately degraded and replaced by a DNA polymerase.</p>
<p>The rationale for this difference is that DNA polymerases have an active site that is geared towards proofreading and that primerless initiation would be an error-prone process. By having the primers 'tagged' by virtue of them being RNA, it is possible for the replication machinery to use them but then replace them with a high fidelity DNA copy of the template strand.</p>
<p><strong>Edit in response to OP comment:</strong>
Synthesis of the leading strand consists of extending an existing DNA. However the leading strand is also originally initiated, at the ori element, with an RNA primer. Once that first initiation event has taken place the synthesis of the leading strand is simply a process of extending that original primer.</p>
<p>Some viruses employ ingenious variations on this theme such as using tRNA primers , or proteins - see <a href="http://en.wikipedia.org/wiki/DNA_replication">Wikipedia</a>.</p>
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https://biology.stackexchange.com/questions/5484/what-is-the-function-of-the-rna-primer-in-dna-replication
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Question: <p>DNA replication goes in the 5' to 3' direction because DNA polymerase acts on the 3'-OH of the existing strand for adding free nucleotides. Is there any biochemical reason why all organisms evolved to go from 5' to 3'? </p>
<p>Are there any energetic/resource advantages to using 5' to 3'? Is using the 3'-OH of the existing strand to attach the phosphate of the free nucleotide more energetically favorable than using the 3'-OH of the free nucleotide to attach the phosphate of the existing strand? Does it take more resources to create a 3' to 5' polymerase?</p>
Answer: <p>Prof. <a href="http://en.wikipedia.org/wiki/User%3aAgathman">Allen Gathman</a> has a great 10-minutes <a href="http://www.youtube.com/watch?v=y4hKibS2fAo">video</a> on Youtube, explaining the reaction of adding nucleotide in the 5' to 3' direction, and why it doesn't work the other way.</p>
<p>Briefly, the energy for the formation of the phosphodiester bond comes from the dNTP, which has to be added. dNTP is a nucleotide which has two additional phosphates attached to its 5' end. In order to join the 3'OH group with the phosphate of the next nucleotide, one oxygen has to be removed from this phosphate group. This oxygen is also attached to two extra phosphates, which are also attached to a Mg++. Mg++ pulls up the electrons of the oxygen, which weakens this bond and the so called nucleophilic attack of the oxygen from the 3'OH succeeds, thus forming the phospodiester bond.</p>
<p>If you try to join the dNTP's 3'OH group to the 5' phosphate of the next nucleotide, there won't be enough energy to weaken the bond between the oxygen connected to the 5' phosphorous (the other two phosphates of the dNTP are on the 5' end, not on the 3' end), which makes the nucleophilic attack harder.</p>
<p>Watch the video, it is better explained there.</p>
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https://biology.stackexchange.com/questions/477/why-is-dna-replication-performed-in-the-5-to-3-direction
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Question: <p><a href="https://www.youtube.com/watch?v=27TxKoFU2Nw" rel="nofollow">https://www.youtube.com/watch?v=27TxKoFU2Nw</a></p>
<p>In the above video it shows that during DNA replication, the lagging strand require RNA primase to add 3' -OH group for further addition of nucleotides. However, it hasn't been shown that the above strand ( leading strand) require it.</p>
<p>Besides, RNA is needed to initiate the polymerization because it has the 3'-OH.
But when I look at the structure of deoxynucleotide, it also has the 3'-OH but it does not have the 2'-OH. So why DNA cannot initiate the polymerization? </p>
<p>Thanks for your answer!</p>
Answer: <p>The <a href="http://en.wikipedia.org/wiki/DNA_polymerase" rel="nofollow noreferrer">DNA polymerase</a> also needs a RNA primer on the leading strand to be able to start polymerization. Afterwards this is not needed anymore, since the <a href="http://en.wikipedia.org/wiki/DNA_replication" rel="nofollow noreferrer">replication</a> goes on without a break. On the lagging strand polymerization replication can only work between the replication fork and the next region of double-stranded DNA. See the figure (from <a href="http://biology.tutorvista.com/cell/dna-replication.html" rel="nofollow noreferrer">here</a>):</p>
<p><img src="https://i.sstatic.net/TeaqU.jpg" alt="enter image description here"></p>
<p>The reason for the need for RNA primers is located in the function of the enzymes. While the DNA polymerase can only work on a double stranded template (add nucleotides to the 3'OH-end of the strand) the <a href="http://en.wikipedia.org/wiki/Primase" rel="nofollow noreferrer">DNA Primase</a> (actually an RNA polymerase) can work on single stranded targets and thus add the RNA primer there.</p>
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https://biology.stackexchange.com/questions/23236/does-dna-replication-in-5-to-3-leading-strand-need-rna-primase
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Question: <p>What is the difference between replication and to divide? My A level bio book says that it takes 20 min for <em>E.coli</em> to divide and in next page it's written that <em>E.coli</em> completes replication within 38min.</p>
<p>Moreover, there is a diagram (shown below) which contradicts as what I thought. </p>
<p>Please explain the difference between replication and division.</p>
<p><a href="https://i.sstatic.net/Sqyqv.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Sqyqv.jpg" alt="enter image description here"></a></p>
Answer: <p>I am unsure if I really understand the question. Are you wondering how the rate of DNA replication can be slower than the rate of cell division if every daughter cell needs a chromosome copy? </p>
<p>If so, keep in mind that prokaryotes have different origins of replication on their chromosome so that <strong>DNA replication can actually be parallelised</strong>. <a href="http://www.sciencedirect.com/science/article/pii/0022283668904257" rel="nofollow">Cooper (1968)</a> presents a mathematical model that is consistent with experimental findings and indicates that when the DNA replication step becomes limiting for cell division, <em>E. coli</em> switches to multiple replication forks, thus decreasing the net time needed for complete DNA replication.</p>
<p>According to that, it is no contradiction that DNA replication <em>per se</em> takes longer than cell division.</p>
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https://biology.stackexchange.com/questions/48112/dna-replication-in-e-coli
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Question: <p>There are many <a href="http://pressbooks-dev.oer.hawaii.edu/biology/chapter/dna-replication-in-eukaryotes/" rel="nofollow noreferrer">statements</a> to be found on the internet of the sort:</p>
<blockquote>
<p>“DNA replication occurs at elongation rates of about 500 nucleotides
per second in bacteria and about 50 nucleotides per second in
vertebrates.”</p>
</blockquote>
<p>However none that I have read attempt to explain this order-of-magnitude difference. Is it known why?</p>
Answer: <p>The difference in DNA replication rate between prokaryotes and eukaryotes is still under current research, but the basics are understood. It is very much a matter of complexity, as eukaryotes are more complex in many different ways. I found a very useful <a href="https://www.nature.com/scitable/topicpage/major-molecular-events-of-dna-replication-413/" rel="nofollow noreferrer">reference</a> for this and other kinds of related questions. Briefly, some possible reasons:</p>
<blockquote>
<p>[...] in eukaryotes, the DNA template is compacted by the way it winds
around proteins called histones. [...]</p>
</blockquote>
<p>The DNA-sequence is not as easily accessible in eukaryotes, the unwrapping and re-wrapping of the DNA takes some time.</p>
<blockquote>
<p>[...] The coordination of the protein complexes required for the steps of
replication and the speed at which replication must occur in order for
cells to divide are impressive, especially considering that enzymes
are also proofreading, which leaves very few errors behind. [...]</p>
</blockquote>
<p>This underlines another rather important factor. <a href="https://bio.libretexts.org/Courses/University_of_California_Davis/BIS_2A%3A_Introductory_Biology_-_Molecules_to_Cell/MASTER_RESOURCES/DNA_Repair_in_Replication" rel="nofollow noreferrer">The speed of the polymerases replicating DNA is very much related to their accuracy.</a></p>
<blockquote>
<p>[...] This proofreading capability comes with some trade-offs: using an
error-correcting/more accurate polymerase requires time (the trade-off
is speed of replication) [...]</p>
</blockquote>
<p>And just for completeness, <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685895/" rel="nofollow noreferrer">here</a> is a compete overview of DNA replication in the three big systems.</p>
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https://biology.stackexchange.com/questions/98150/why-is-dna-replication-so-much-faster-in-prokaryotes-than-eukaryotes
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Question: <p><span class="math-container">$ E.coli $</span> has circular DNA which I guess implies one strand forms the outer circle and the other the inner one. So, is there a way to know if the replicated DNA forms the outer or inner circle?
In the image attached it is seen that the replicated DNA forms the inner circle. Also mentioned is the fact that it is not possible to make out individual strands by TEM.<img src="https://i.sstatic.net/ZhxjU.jpg" alt="enter image description here"><img src="https://i.sstatic.net/9YDHe.jpg" alt="enter image description here"></p>
Answer:
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https://biology.stackexchange.com/questions/82886/dna-replication-in-e-coli
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Question: <p><img src="https://i.sstatic.net/khA5P.png" alt="Unrepaired Mistakes in DNA Synthesis Lead to Point Mutations"></p>
<p>Hi! I'm trying to make sense of this illustration (from the textbook Biological Science by Scott Freeman).</p>
<p>The general question is: How do point mutations arise from mistakes in DNA replication?</p>
<p>If you don't mind, however, I'd like to explain how I interpret the illustration so you can see the confusion. As the original molecule of DNA is replicated (in gray), a mistake occurs in the synthesis of the bottom strand in the new molecule (the noncomplimentary bases G and T have been paired together). Now, it seems like a second replication is required for the mutation to arise: the defective molecule is replicated resulting in two new molecules, one free of mistakes (because it takes as its template the top strand) and one "wrong" where the mutation is present. </p>
<p>But my doubt is, wouldn't the middle molecule already constitute a mutation? If a mRNA were to transcribe that sequence, the codon is already different from the original molecule. Must a DNA molecule be replicated two times for a mutation to arise (where the first time a mistake is made and a second where such mistake is, let's say, "consolidated")?</p>
<p>Thank you very much in advance.</p>
Answer: <p><strong>Source of your misunderstanding</strong></p>
<p>Your misunderstanding is very comprehensible as the figure is misleading.</p>
<p>The figure only shows a single event of replication. What you see as a second replication resulting into two double stranded molecules is NOT an event of replication. It actually represents the two possible outcomes from a 'mismatch repair mechanism'. The term <code>DNA replication</code> written on the figure should be replaced by <code>Possible outcomes of DNA repair</code>. The molecule that contains the <code>G-T</code> mismatch is therefore just a temporary state that will very quickly be changed to either the <code>T-A</code> state (bottom right of your figure) or to the <code>G-C</code> state (upper right of your figure). More information below.</p>
<p><strong>DNA repair</strong></p>
<blockquote>
<p><a href="https://en.wikipedia.org/wiki/DNA_repair" rel="nofollow noreferrer">DNA repair</a> is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome.</p>
</blockquote>
<p>The type of DNA repair that is of interest on the figure is called "DNA mismatch repair"</p>
<p><strong>DNA mismatch repair</strong></p>
<blockquote>
<p><a href="https://en.wikipedia.org/wiki/DNA_mismatch_repair" rel="nofollow noreferrer">DNA mismatch repair</a> is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.</p>
</blockquote>
<p>There are specific enzymes to repair a mismatch such as the <code>G-T</code> mismatch represented on your figure. These enzymes can either repair <code>G-T</code> into <code>G-C</code> or into <code>A-T</code>. If the repair is <code>A-T</code> (lower outcome in the figure) then a mutation would have occurred. If the repair is <code>G-C</code> (upper outcome in the figure) then we are back to the original sequence and no mutation would have occurred.</p>
<p>Note that the probability of the two possible outcomes is different from 0.5 as these enzymes have ways to try to figure out which was the original strand and which was the newly replicated strand. You can learn much more about the mechanism of DNA mismatch repair on the <a href="https://en.wikipedia.org/wiki/DNA_mismatch_repair" rel="nofollow noreferrer">wikipedia > DNA mismatch repair</a>.</p>
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https://biology.stackexchange.com/questions/55364/how-do-point-mutations-arise-from-mistakes-in-dna-replication
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Question: <p><a href="https://i.sstatic.net/79S08.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/79S08.png" alt="enter image description here"></a></p>
<p>Hi there,</p>
<p>I am confused about how the nucleophilic attack occurs in DNA replication.I watched this video from a biology professor (<a href="https://www.youtube.com/watch?v=y4hKibS2fAo" rel="nofollow noreferrer">https://www.youtube.com/watch?v=y4hKibS2fAo</a>)</p>
<p>I watched this video which stated that during DNA replication, the hydroxl group forms a covalent bond with the phosphate and the two phosphorus groups are a leaving group.</p>
<p>I understand the basics of these kinds of reactions, but this one just seems confusing. I can see that the oxygen which was part of the hydroxl group forms a covalent bond with phosphorus. But what happens to the hydrogen? And why does the the double bond and position of the HO change position from the first picture to the second picture? </p>
<p>There are also some pictures which show water being produced. But the video states that the hydroxl group is forming a bond with the phosphate group. How does that work?</p>
<p>Thanks a lot</p>
Answer: <p>Polymerases uses a two-metal ion mechanism to carry out the addition of a new NTP to a growing RNA strand. Two Mg2+ ions are used in this process; metal ion A is involved in the formation of the nucleophile (O-) for the SN2 reaction to occur and metal ion B is involved in the stabilization of the transition state in the reaction. For the addition of a NTP to a RNA strand to occur, an activated 3’ hydroxyl group (O-) acts a nucleophile and attacks the alpha phosphate of the incoming NTP. The reaction is initiated by a deprotonation event. Metal ion A aids with this deprotonation as it reduces the pKa of the hydroxyl (so it is more acidic), making it easier to deprotonate and form the nucleophile, the proton is accepted by a nearby aspartate residue (<strong>not water</strong>) - doi: 10.1021/ja403842j. After the hydroxyl attack, the mechanism proceeds through a pentacovalent transition state like in a standard Sn2 reaction and the pyrophosphate group (PPi) is kicked out. The image is not mine but comes from this paper: <a href="https://doi.org/10.1038/34542" rel="nofollow noreferrer">https://doi.org/10.1038/34542</a>, which is quite good, as is this paper: doi: 10.1074/jbc.274.25.17395 and this one <a href="https://doi.org/10.1016/j.molcel.2006.03.013" rel="nofollow noreferrer">https://doi.org/10.1016/j.molcel.2006.03.013</a>
<a href="https://i.sstatic.net/09Uq8.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/09Uq8.jpg" alt="enter image description here"></a></p>
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https://biology.stackexchange.com/questions/82345/how-does-the-nucleophilic-attack-in-dna-replication-occur
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Question: <p>this is my first time here, so go easy on me! I've been trying to find out more about the actual process of DNA replication. Specifically, I am wondering if, when the DNA replicates during cellular division, the result is the <em>original</em> DNA strand and a copy? Or is the original strand destroyed in the process and there are 2 child copies that are identical (aside from mutations) to the original?</p>
<p>I am pretty confident in the end-result, but maybe whoever answers can verify this for me too. My understanding is that the original cell itself is destroyed in the end, but the daughter cells are constructed from the substance of the parent's cell. The initial question will help clarify this process for me too, because I am curious if one of the two daughter cells has the literal DNA from the original, or if basically the entire original cell "dies" in the process and the two new cells are completely new.</p>
<p><strong>Update</strong> (to demonstrate research effort):</p>
<p>I have learned how a helicase enzyme untwists the DNA and severs the hydrogen bonds between the nucleotide bases, then single-strand binding proteins bind DNA polymerase to the ends of the strand and begin molding, creating a complimentary strand for each side. It seems evident that half of the "original" is found in each daughter, and the DNA polymerase forms the other half. So, the original is still present, but not in its original form, which is why I'm not sure what the "official" diagnosis is. Is it a parent-child, or two children?</p>
Answer: <p>Via: <a href="https://en.wikipedia.org/wiki/Semiconservative_replication" rel="nofollow noreferrer">https://en.wikipedia.org/wiki/Semiconservative_replication</a></p>
<blockquote>
<p><strong>Semiconservative replication</strong> describes the mechanism of DNA replication in all known cells. It derives its name from the fact that it produces two copies of the original DNA molecule, each of which contains <em>one of original strand</em>, and <em>one newly-synthesized strand</em>. (emph. added)</p>
</blockquote>
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https://biology.stackexchange.com/questions/85435/dna-replication-2-new-strands-or-original-parent-and-child
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Question: <p><a href="https://www.youtube.com/watch?v=8kK2zwjRV0M" rel="nofollow noreferrer">This video</a> entertainingly supplements this <a href="https://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html" rel="nofollow noreferrer">3d animation</a> of DNA strand replication. Does this process happen serially from the beginning to the end of a DNA strand (like having to unzip a six foot long zipper - example A below) or does the strand get chopped up into chunks, unzipped individually, replicated then brought back together (example B)? </p>
<p><a href="https://i.sstatic.net/Tkrfv.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Tkrfv.png" alt="enter image description here"></a></p>
<p>I suspect the answer is case A. If so, is any phase of replication done in a parallel fashion? I would think something has to be (since it happens so fast and serial anything is typically very slow in comparison).</p>
Answer:
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https://biology.stackexchange.com/questions/58728/is-dna-replication-a-serial-or-parallel-process
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Question: <p>In a particular strain of <em>E. coli</em>, it was observed that DNA polymerase could add nucleotides to a growing chain of DNA at the rate of 600 per second. If the genome of this organism is 1.1mm long wherein a base pair occupies 0.34 nm, then how much time (in minutes) would be required for the complete replication of the chromosomal DNA molecule?
(Report the closest integer value.) {<a href="https://olympiads.hbcse.tifr.res.in/olympiads/wp-content/uploads/2016/09/inbo2014-Q.pdf" rel="nofollow noreferrer">Source</a>}</p>
<p>I solved this question using this method.</p>
<p>No. of base pairs: <span class="math-container">$ \frac{1.1 \times 10^{-3} \space m}{0.34 \times 10^{-9} \space m/bp} = 3.23 \times 10^{6} \space bp$</span></p>
<p>Time required for replication (in minutes by the DNA Polymerase) = <span class="math-container">$ \frac{ 3.23 \times 10^{6} \space bp}{(600 \space bp/s)\times (60 \space s/minute)} =89.86 ≈ 90 \space minutes$</span></p>
<p>The answer, however, is 45 minutes. What am I getting wrong?</p>
Answer: <p>Remember that <em>E. coli</em> has a single circular chromosome, and that chromosome is replicated bidirectionally. Hence, your calculated value (90 minutes) is exactly twice that of the correct answer.</p>
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https://biology.stackexchange.com/questions/107396/time-required-for-dna-replication-in-e-coli
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Question: <p>An article in <em>Nature Scitable</em> on <a href="https://www.nature.com/scitable/topicpage/dna-replication-and-causes-of-mutation-409" rel="nofollow noreferrer">DNA Replication and the causes of Mutation</a> states that:</p>
<blockquote>
<p>When an incorrect nucleotide is added to the growing strand, replication is stalled by the fact that the nucleotide's exposed 3′-OH group is in the "wrong" position… During proofreading, DNA polymerase enzymes recognize this and replace the incorrectly inserted nucleotide so that replication can continue. <strong>Proofreading fixes about 99% of these types of errors</strong></p>
</blockquote>
<p>I am interested to know how this value of 99% was determined, and this is not explained in the article.</p>
Answer:
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https://biology.stackexchange.com/questions/71017/how-is-the-effect-of-proof-reading-on-error-frequency-during-dna-replication-det
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Question: <p>DNA polymerases have proof-reading ability, but RNA polymerase does not. Does the use of RNA as a primer affect the accuracy of DNA replication in E.coli? Explain</p>
Answer: <p>E.coli use <strong>DNA polymerase</strong> for DNA replication too. <strong>Primase</strong> creates a short oligonucleotid (<strong>primer</strong>) in the start of string and DNA polymerase continues in work. <strong>RNA polymerase</strong> is using in synthesis of RNA molecules.</p>
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https://biology.stackexchange.com/questions/45512/does-the-use-of-rna-as-a-primer-affect-the-accuracy-of-dna-replication-in-e-coli
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Question: <p>The lagging strand, downstream of the Okazaki fragment, is covered in single-stranded binding proteins (SSBPs) during DNA replication. What is the mechanism which ensures that SSBPs are removed from the lagging strand to allow for the binding of the next Okazaki fragment?</p>
Answer: <p>Actually, there has been some evidence that the SSBPs keep the bases facing outwards so DNA polymerase can still replicate the lagging strand with the SSBPs on them. There has also been other evidence that they can pop off spontaneously.</p>
<p>You can learn more about this by watching the lectures on <a href="https://www.edx.org/course/molecular-biology-part-1-dna-replication-and-repair" rel="nofollow noreferrer">https://www.edx.org/course/molecular-biology-part-1-dna-replication-and-repair</a></p>
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https://biology.stackexchange.com/questions/72761/how-are-single-stranded-binding-proteins-removed-from-the-lagging-strand-during
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Question: <p>I understand multiple origin bubbles; DNA polymerase only synthesizes DNA from 5' to 3' and all that. But what I don't understand is why it has to be in fragments. Yes, DNA is anti parallel, and so the DNA elongates in opposite directions, since DNA polymerase can only go one way. But why not go on just like the leading strand? Why not continue happily along the DNA template continuously but in fragments? </p>
Answer: <p>I think you may have been misled by graphic representations of the process: The actual replication fork is very small as, like Rex Kerr mentions, it costs a lot of energy to keep DNA single stranded. </p>
<p>Have a watch of <a href="https://www.youtube.com/watch?v=yqESR7E4b_8" rel="nofollow">https://www.youtube.com/watch?v=yqESR7E4b_8</a> at minute 1:45, it contains a relatively realistic representation of the replication fork. Of course you have to imagine all of the space around the molecules in this video to be filled with other DNA, RNA, proteins and small molecules zipping about.</p>
<p>On top of this, we like to think of DNA as a lone double strand where occasionally a protein might attach and do something. Realistically however, most of the DNA is constantly being manipulated by all sorts of proteins (mostly histones for compression, but also transcription factors, methylating and demethylating enzymes, DNA repair enzymes,...). DNA replication has to minimise the interruption it causes to all the mechanisms going on around it, and the most effective way to do this is by replicating the lagging strands in fragments which can be zipped up quickly and left to whatever other proteins might do to it.</p>
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https://biology.stackexchange.com/questions/8305/dna-replication-okazaki-fragments
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Question: <blockquote>
<p>"Each gamete is genetically unique because the DNA of the parent cell is shuffled before the cell divides. This helps ensure that the new organisms formed as a result of sexual reproduction are also unique."</p>
</blockquote>
<p>Then why do we say that the DNA of the parent influences the characteristics of the child while the DNA of the child is formed as a combination of shuffled up nitrogenous and phosphate bases?</p>
Answer: <p>To explain it briefly: </p>
<p>Lets take a human as example, you are diploid and you have a pair of 23 chromosomes (= total 46) and the sex chromosomes which I will exclude for this explanation. </p>
<p>Your gamete is haploid and has therefore only one of the two paired chromosomes. So for every chromosome pair there are 2 possible chromosomes. So in total you have 2<sup>23</sup> possibilites to arrange chromosomes. The gamete from the sexual mating partner also has 2<sup>23</sup> possibilites to arrange chromosomes. So in total a new diploid organism has (2<sup>23</sup>)x(2<sup>23</sup>) possibilites. So unique. </p>
<p>However, it is always the chromosomes of the parents. So yes, parents influence the children because the genetic information of the children can be found in one of the two parents. </p>
<p>Furthermore you have recombination, but you already asked another question about this. So I will not expand this answer. </p>
<p>There are relevant articles in Wikipedia on <a href="https://en.wikipedia.org/wiki/Genetic_recombination" rel="nofollow noreferrer">genetic recombination</a> and <a href="https://en.wikipedia.org/wiki/Meiosis" rel="nofollow noreferrer">meiosis</a> that I would recommend.</p>
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https://biology.stackexchange.com/questions/68970/dna-replication-and-combination
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Question: <p>I am studying a paper about the relation between polyP granule and cell cycle exit. As the author explained the four general steps for cell cycle exit, the second step is" the completion of open rounds of DNA replication". But I really can't figure out the meaning of this sentence. I hope that someone can explain this to me. Thank you! </p>
Answer: <p>From the context of the <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373386/" rel="nofollow noreferrer">article I found from an internet search</a>:</p>
<blockquote>
<p>Cell cycle exit in bacteria encompass four general steps:</p>
<p>(i) inhibition of inappropriate reinitiation of DNA replication,</p>
<p>(ii) <strong>completion of open rounds of DNA replication</strong>,</p>
<p>(iii) segregation and compaction of daughter chromosomes, and</p>
<p>(iv) septation.</p>
</blockquote>
<p>I would assume that ‘open rounds’ means ‘DNA replication that has already been started’ (‘open’ signifying already started, and a ‘round’ has the sense of one event of a series). ‘Completion’ is obviously ‘finishing’, ‘ending’ — the cell cycle doesn’t end until all the replication that has started has finished.</p>
<p>I have not seen the adjective ‘open’ used in this way previously, and suspect it is uncommon, but as this is not my field I stand to be corrected.</p>
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https://biology.stackexchange.com/questions/79933/what-is-the-meaning-of-the-following-completion-of-open-rounds-of-dna-replica
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Question: <p>I know that when two sugar molecules (like glucose) connect to each other, H<sub>2</sub>O is released because of the -OH and -H groups in both of the molecules. I want to know if the same thing happens when two nucleotides connect to each other during DNA replication.</p>
Answer: <p>yes there is no water release during phosphodiester linkages because the 3'OH of the growing daughter strand exerts a nucleophilic attack on the phosphodiester linkage between the alpha phosphate with the beta & gamma phoshate of the deoxyribonucleoside triphosphate.</p>
<p>During such formation there is no hydrolysis rather it would precisely be a transesterification type of organic reaction.</p>
<p>REFERENCES:TEXT BOOK OF BIOCHEMISTRY(T.M. DEVLIN)
<a href="https://en.m.wikipedia.org/wiki/Transesterification" rel="nofollow noreferrer">https://en.m.wikipedia.org/wiki/Transesterification</a></p>
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https://biology.stackexchange.com/questions/61319/is-water-released-when-a-phosphodiester-bond-is-made-between-two-nucleotides-dur
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