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Designing antiviral therapeutics is of great concern per current pandemics caused by novel coronavirus or SARS-CoV-2. The core polymerase enzyme in the viral replication/transcription machinery is generally conserved and serves well for drug target. In this work we briefly review structural biology and computational clues on representative single-subunit viral polymerases that are more or less connected with SARS-CoV-2 RNA dependent RNA polymerase (RdRp), in particular, to elucidate how nucleotide substrates and potential drug analogs are selected in the viral genome synthesis. To do that, we first survey two well studied RdRps from Polio virus and hepatitis C virus in regard to structural motifs and key residues that have been identified for the nucleotide selectivity. Then we focus on related structural and biochemical characteristics discovered for the SARS-CoV-2 RdRp. To further compare, we summarize what we have learned computationally from phage T7 RNA polymerase (RNAP) on its stepwise nucleotide selectivity, and extend discussion to a structurally similar human mitochondria RNAP, which deserves special attention as it cannot be adversely affected by antiviral treatments. We also include viral phi29 DNA polymerase for comparison, which has both helicase and proofreading activities on top of nucleotide selectivity for replication fidelity control. The helicase and proofreading functions are achieved by protein components in addition to RdRp in the coronavirus replication-transcription machine, with the proofreading strategy important for the fidelity control in synthesizing a comparatively large viral genome.

RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and fidelity control in the RNAP transcription elongation. Here we summarize our computational work from several recent publications to demonstrate first how T7 RNAP translocates via Brownian alike motions along DNA right after the catalytic product release. Then we show how the backward translocation motions are prevented at post-translocation upon successful nucleotide incorporation, which is also subject to stepwise nucleotide selection and acts as a pawl for "selective ratcheting". The structural dynamics and energetics features revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet working scenario of a prototypical transcription machine with sophisticated nucleotide selectivity for fidelity control. The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPs and some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machinery in higher organisms.

High fidelity gene transcription and replication require kinetic discrimination of nucleotide substrate species by RNA and DNA polymerases under chemical non-equilibrium conditions. It is known that sufficiently large free energy driving force is needed for each polymerization or elongation cycle to maintain far-from-equilibrium to achieve low error rates. Considering that each cycle consists of multiple kinetic steps with different transition rates, one expects that the kinetic modulations by polymerases are not evenly conducted at each step. We show that accelerations at different kinetic steps impact quite differently to the overall elongation characteristics. In particular, for forward transitions that discriminate cognate and non-cognate nucleotide species to serve as kinetic selection checkpoints, the transition cannot be accelerated too quickly nor retained too slowly to obtain low error rates, as balancing is needed between the nucleotide selectivity and the non-equilibrium driving. Such a balance is not the same as the speed-accuracy tradeoff in which high accuracy is always obtained at sacrifice of speed. For illustration purposes, we used three-state and five-state models of nucleotide addition in the polymerase elongation and show how the non-equilibrium steady state characteristics change upon variations on stepwise forward or backward kinetics. Notably, by using the multi-step elongation schemes and parameters from T7 RNA polymerase transcription elongation, we demonstrate that individual transitions serving as selection checkpoints need to proceed at moderate rates in order to sustain the necessary non-equilibrium drives as well as to allow nucleotide selections for an optimal error control. We also illustrate why rate-limiting conformational transitions of the enzyme likely play a significant role in the error reduction.

Designing antiviral therapeutics is of great concern per current pandemics caused by novel coronavirus or SARS-CoV-2. The core polymerase enzyme in the viral replication/transcription machinery is generally conserved and serves well for drug target. In this work we briefly review structural biology and computational clues on representative single-subunit viral polymerases that are more or less connected with SARS-CoV-2 RNA dependent RNA polymerase (RdRp), in particular, to elucidate how nucleotide substrates and potential drug analogs are selected in the viral genome synthesis. To do that, we first survey two well studied RdRps from Polio virus and hepatitis C virus in regard to structural motifs and key residues that have been identified for the nucleotide selectivity. Then we focus on related structural and biochemical characteristics discovered for the SARS-CoV-2 RdRp. To further compare, we summarize what we have learned computationally from phage T7 RNA polymerase (RNAP) on its stepwise nucleotide selectivity, and extend discussion to a structurally similar human mitochondria RNAP, which deserves special attention as it cannot be adversely affected by antiviral treatments. We also include viral phi29 DNA polymerase for comparison, which has both helicase and proofreading activities on top of nucleotide selectivity for replication fidelity control. The helicase and proofreading functions are achieved by protein components in addition to RdRp in the coronavirus replication-transcription machine, with the proofreading strategy important for the fidelity control in synthesizing a comparatively large viral genome.

Polymerases select nucleotides according to a template before incorporating them for chemical synthesis during gene replication or transcription. Efficient selection to achieve sufficiently high fidelity and speed is essential for polymerase function. Due to multiple kinetic steps detected in a polymerase elongation cycle, there exist multiple selection checkpoints to allow different strategies of fidelity control. In our current work, we examined step-by-step selections in an elongation cycle that have conformational transition rates tuned one at a time, with a controlled differentiation free energy between the right and wrong nucleotides at each checkpoint. The elongation is sustained at non-equilibrium steady state with constant free energy input and heat dissipation. It is found that a selection checkpoint in the later stage of a reaction path has less capability for error reduction. Hence, early selection is essential to achieve an efficient fidelity control. In particular, for an intermediate state, the selection through the forward transition inhibition has the same capacity for error reduction as the selection through the backward rejection. As with respect to the elongation speed, an initial screening is indispensible for maintaining high speed, as the wrong nucleotides can be removed quickly and replaced by the right ones at the entry. Overall, the elongation error rate can be repeatedly reduced through multiple selection checkpoints. This study provides a theoretical framework to guide more detailed structural dynamics studies, and to support rational redesign of related enzymes and devices.

High fidelity gene transcription and replication require kinetic discrimination of nucleotide substrate species by RNA and DNA polymerases under chemical non-equilibrium conditions. It is known that sufficiently large free energy driving force is needed for each polymerization or elongation cycle to maintain far-from-equilibrium to achieve low error rates. Considering that each cycle consists of multiple kinetic steps with different transition rates, one expects that the kinetic modulations by polymerases are not evenly conducted at each step. We show that accelerations at different kinetic steps impact quite differently to the overall elongation characteristics. In particular, for forward transitions that discriminate cognate and non-cognate nucleotide species to serve as kinetic selection checkpoints, the transition cannot be accelerated too quickly nor retained too slowly to obtain low error rates, as balancing is needed between the nucleotide selectivity and the non-equilibrium driving. Such a balance is not the same as the speed-accuracy tradeoff in which high accuracy is always obtained at sacrifice of speed. For illustration purposes, we used three-state and five-state models of nucleotide addition in the polymerase elongation and show how the non-equilibrium steady state characteristics change upon variations on stepwise forward or backward kinetics. Notably, by using the multi-step elongation schemes and parameters from T7 RNA polymerase transcription elongation, we demonstrate that individual transitions serving as selection checkpoints need to proceed at moderate rates in order to sustain the necessary non-equilibrium drives as well as to allow nucleotide selections for an optimal error control. We also illustrate why rate-limiting conformational transitions of the enzyme likely play a significant role in the error reduction.

Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensively characterized. Using hydrogen-deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2-furfuryl-deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6-methyl-deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates. [ABSTRACT FROM AUTHOR]

Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2′-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases. [ABSTRACT FROM AUTHOR]

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the 3-phosphate and base of the matched nucleotide triphosphate (NIP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild- type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu108l, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity. [ABSTRACT FROM AUTHOR]

Abstract: Positive-strand RNA viruses exist as a quasi-species due to the incorporation of mutations into the viral genome during replication by the virus-encoded RNA-dependent RNA polymerase (RdRP). Therefore, the RdRP is often described as a low-fidelity enzyme. However, until recently, a complete description of the kinetic, thermodynamic and structural basis for the nucleotide incorporation fidelity of the RdRP has not been available. In this article, we review the following: (i) the steps employed by the RdRP to incorporate a correct nucleotide; (ii) the steps that are employed by the RdRP for nucleotide selection; (iii) the structure-based hypothesis for nucleotide selection; (iv) the impact of sites remote from the active site on polymerase fidelity. Given the recent observation that RNA viruses exist on the threshold of error catastrophe, the studies reviewed herein suggest novel strategies to perturb RdRP fidelity that may lead ultimately to the development of antiviral agents to treat RNA virus infection. [Copyright &y& Elsevier]

RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and fidelity control in the RNAP transcription elongation. Here we summarize our computational work from several recent publications to demonstrate first how T7 RNAP translocates via Brownian alike motions along DNA right after the catalytic product release. Then we show how the backward translocation motions are prevented at post-translocation upon successful nucleotide incorporation, which is also subject to stepwise nucleotide selection and acts as a pawl for "selective ratcheting". The structural dynamics and energetics features revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet working scenario of a prototypical transcription machine with sophisticated nucleotide selectivity for fidelity control. The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPs and some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machinery in higher organisms.