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I'm doing a presentation on the replication of SARS-CoV-2 for my chemistry class, and I found that to replicate its RNA, the virus uses RNA-dependent RNA polymerase, which is primed by a VPg primer. Since the presentation is for a chemistry class, not a molecular biology class, I would like to know the chemistry behind the way that primers work.

I've tried searching the information on the internet, but could not locate the information I need.

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    $\begingroup$ Can you clarify what it is that you are looking for? What is it that you need that resources like the Wikipedia page on base pairing do not provide? $\endgroup$ – acvill Jun 6 '20 at 23:59
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    $\begingroup$ Welcome to Biology.SE! We expect you to do research on your own and then, informed by what you have learned, ask any questions you still have (ideally with references to reliable sources). For "homework" questions, you are required to show your attempt to answer the question and to use the "homework" tag. This wikipedia page may be a helpful starting point. ——— Please take the tour and then go through the help pages starting with How to Ask questions effectively on this site. Thanks! 😊 $\endgroup$ – tyersome Jun 7 '20 at 0:36
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    $\begingroup$ I have edited your title so that it is clear you are talking about the specific VPg primer for the replication of RNA viruses like coronoviruses. It would helpful if you could tell what you know about the chemistry of RNA polymerase already so that I (as an ex-chemist) can an answer your question appropriately from a chemical standpoint. $\endgroup$ – David Jun 8 '20 at 10:09
  • $\begingroup$ Search for Subissi papers on the CoV RdRp, he did several in-vitro experiments, mixing directly some nucleosides, oligos, nsp7,8 and 12 in a test tube and looking at what was synthesized. pubmed.ncbi.nlm.nih.gov/25197083 $\endgroup$ – reuns Jul 10 '20 at 17:03
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Although this question should probably be ruled off-topic as unresearched homework, I fancy that not all readers will be familiar with the VPg primer for viral RNA replication. I shall therefore suggest some of the chemical aspects of this process that might be include in a presentation, and comment on the process itself.

Some Chemical features of VPg-primed viral RNA replication

  1. The chemical reaction. This is no different from many other reactions involving an RNA polymerase. It involves the reaction of an NTP with the 3’OH of the ribose on a growing RNA chain, forming a phosphodiester bond, thus extending the chain. The growing RNA chain is termed a primer because it is required to start the addition in many cases, however in the case of many single-stranded RNA viruses the actual initiation of a new strand uses a primer composed of a small peptide to which to U residues have been attached. Simple diagrams of this process are generally presented for DNA-dependant DNA polymerase and can be found in standard texts, e.g. Berg et al. Figure 5.22

  2. The reaction mechanism. This reaction is catalysed by an enzyme which participates in the reaction. The mechanism of catalysis is summarized on the EBI facility (where diagrams can be found) as:

RNA polymerases catalyse the nucleophilic attack of a bound nucleoside 5′-triphosphate by the 3′-hydroxyl of an RNA primer, resulting in the incorporation of a nucleoside monophosphate into RNA and the release of pyrophosphate. This is thought to occur using two-metal catalysis. In RNA polymerase II, two magnesium ions are coordinated by four aspartates (3′OH of RNA also proposed to weakly coordinate to Mg2+A). Mg2+A is proposed to lower the pKa around the attacking hydroxyl while Mg2+B is there to stabilise the negative charges during transition state…

  1. The thermodynamics of the reaction. The breaking of one phosphodiester bond and the formation of another means that the reaction is more or less at equilibrium (no change in Gibbs Free Energy). One reason that it is thought not to reverse is because the pyrophosphate produced is hydrolysed in the cell to orthophosphate, which is irreversible as the free energy change in this reaction is lost as heat.

  2. Hydrogen Bonding to the template. The reaction involves copying a template strand, and the basis of this specificity is Watson–Crick hydrogen bonding between template base and incoming NTP. However, specifically for the VPg primer, there is hydrogen bonding of the two U residues to the end of the template strand. (Diagrams of AU hydrogen bonding should be easy to find.)

  3. The chemical linkage of the VPgUU primer. The uridylic acid residues are added to the VPg peptide at the hydroxyl group of a tyrosine residue in a reaction that is catalysed by the viral RNA polymerase. It is worth noting that although many amino acid residues of proteins are chemically inert, the hydroxy amino acids have reactive potential, most notably in being phosphorylated.

VPgUU

A broader point for reflection

One question that this raises is why the need for a protein — VPg — as adjunct to a nucleic acid primer. Reading further on the subject will reveal this protein actually interacts with different parts of the viral RNA in the course of acquiring the Uridylate residues and selecting the AA where it should bind. The chemical interactions with RNA and other proteins here are the weak non-covalent interactions — hydrogen bonds, van der Walls interactions and some ionic interactions — that are typical of proteins. Rather than their weakness being a reason for considering them unimportant, such interactions are crucial to the chemistry of life because their very weakness means that they can be both broken as well as made. The interactions are reversible, leading to the very dynamic property that typifies life. They are worthy of a chemist’s attention.

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Nucleic acid synthesys can occur two ways, with a template or without. In most biological systems the template synthesis is the one that occurs since it is the only way to replicate genomic information. For template based nucleic acid synthesis in biological systems (being of DNA or RNA) the enzymes performing the reactions can use single stranded material (in the case of virus ssDNA or ssRNA virus) however they still need a small region of double stranded nucleic acid to bind and start polymerizing, otherwise synthesis could start anywhere in the template strand which wouldn't be particulartly useful, hence the primers, the polymerases bind to the double stranded region and start polymerizing always adding new nucleotides to the 3' end of the primer strand that are complementary to the nucleotide in the template strand. Usually in living cells the synthesis of new RNA molecules won't occur with an RNA template (there is usually no RNA dependent RNA polymerase, there are exceptions but they are usually result of previous viral infections), but only with a DNA template to procuce messenger/transfer/ribossomal RNAs. In the case of COVID and othe + strand RNA virus, the virus has to encode the RNA polymerase and also produce a primer that will create a double stranded region that will allow RNA synthesis to occur.

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    $\begingroup$ Hello @lint, and welcome to Biology.SE! Yours is a good explanation of the reasons for using primers and their importance in guiding polymerase amplification. You clearly explain the molecular biology of PCR, but OP is explicitly looking for a chemistry perspective -- perhaps one that includes a discussion of the hydrogen bonding involved in primer specificity and the catalysis of DNA polymerization. $\endgroup$ – acvill Jun 7 '20 at 19:12
  • $\begingroup$ I don't think there are any biochemical reasons for the requirement of primers. The need for a primer in the synthesis relates to the requirement ofhaving a double stranded region that guides where in the sequence the process starts, otherwise you would have an entire population of molecules with different sizes and start sites. There are cases where primers are not needed for nucleic acid synthesis from template like RNA transcription and DNA replication start, in this case the specificity of the start is given by the recognition of specific sequences by the machinery. $\endgroup$ – lint Jun 7 '20 at 21:20
  • $\begingroup$ @lint — The need for a primer is tied up with ensuring the ends of a linear genome get replicated. Easier than for a double-stranded chromosome, but still an important consideration. It's not that there will be a population with different sizes of RNA that is important, it's that the genome would get shorter and shorter. $\endgroup$ – David Jun 8 '20 at 18:01

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