Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers.
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ABSTRACT:
SUBMITTER: Dulin D
PROVIDER: S-EPMC5670035 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous 'synthetic nucleic acid substitution' strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-'substituted' NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the β,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.
| S-EPMC4857003 | biostudies-literature
Project description:Eukaryotes express three or more multisubunit nuclear RNA polymerases (Pols) referred to as Pols I, II, and III, each of which synthesizes a specific subset of RNAs. Consistent with the diversity of their target genes, eukaryotic cells have evolved divergent cohorts of transcription factors and enzymatic properties for each RNA polymerase system. Over the years, many trans-acting factors that orchestrate transcription by the individual Pols have been described; however, little effort has been devoted to characterizing the molecular mechanisms of Pol I activity. To begin to address this gap in our understanding of eukaryotic gene expression, here we establish transient-state kinetic approaches to characterize the nucleotide incorporation mechanism of Pol I. We collected time courses for single turnover nucleotide incorporation reactions over a range of substrate ATP concentrations that provide information on both Pol I's nucleotide addition and nuclease activities. The data were analyzed by model-independent and model-dependent approaches, resulting in, to our knowledge, the first minimal model for the nucleotide addition pathway for Pol I. Using a grid searching approach we provide rigorous bounds on estimated values of the individual elementary rate constants within the proposed model. This work reports the most detailed analysis of Pol I mechanism to date. Furthermore, in addition to their use in transient state kinetic analyses, the computational approaches applied here are broadly applicable to global optimization problems.
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Project description:DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative representative of a severe respiratory illness resulted in widespread human infections and deaths in nearly all of the countries since late 2019. There is no therapeutic FDA-approved drug against SARS-CoV-2 infection, although a combination of anti-viral drugs is directly being practiced in some countries. A broad-spectrum of antiviral agents are being currently evaluated in clinical trials, and in this review, we specifically focus on the application of Remdesivir (RVD) as a potential anti-viral compound against Middle East respiratory syndrome (MERS) -CoV, SARS-CoV and SARS-CoV-2. First, we overview the general information about SARS-CoV-2, followed by application of RDV as a nucleotide analogue which can potentially inhibits RNA-dependent RNA polymerase of COVs. Afterwards, we discussed the kinetics of SARS- or MERS-CoV proliferation in animal models which is significantly different compared to that in humans. Finally, some ongoing challenges and future perspective on the application of RDV either alone or in combination with other anti-viral agents against CoVs infection were surveyed to determine the efficiency of RDV in preclinical trials. As a result, this paper provides crucial evidence of the potency of RDV to prevent SARS-CoV-2 infections.Communicated by Ramaswamy H. Sarma.
| S-EPMC7256352 | biostudies-literature
Project description:All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called "cleavage factors" appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase's cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase-cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases.
| S-EPMC6400273 | biostudies-literature
Project description:All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.
| S-EPMC3820909 | biostudies-literature
Project description:RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage ?6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that ?6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.
| S-EPMC7261197 | biostudies-literature
Project description:There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5'-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5'-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5'-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5'-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104 This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.
| S-EPMC5786792 | biostudies-literature
Project description:Poliovirus (PV) is a well-characterized RNA virus, and the RNA-dependent RNA polymerase (RdRp) from PV (3D(pol)) has been widely employed as an important model for understanding the structure-function relationships of RNA and DNA polymerases. Many experimental studies of the kinetics of nucleotide incorporation by RNA and DNA polymerases suggest that each nucleotide incorporation cycle basically consists of six sequential steps: (1) an incoming nucleotide binds to the polymerase-primer/template complex; (2) the ternary complex (nucleotide-polymerase-primer/template) undergoes a conformational change; (3) phosphoryl transfer occurs (the chemistry step); (4) a post-chemistry conformational change occurs; (5) pyrophosphate is released; (6) RNA or DNA translocation. Recently, the importance of structural motif D in nucleotide incorporation has been recognized, but the functions of motif D are less well explored so far. In this work, we used two computational techniques, molecular dynamics (MD) simulation and quantum mechanics (QM) method, to explore the roles of motif D in nucleotide incorporation catalyzed by PV 3D(pol). We discovered that the motif D, exhibiting high flexibility in either the presence or the absence of RNA primer/template, might facilitate the transportation of incoming nucleotide or outgoing pyrophosphate. We observed that the dynamic behavior of motif A, which should be essential to the polymerase function, was greatly affected by the motions of motif D. In the end, through QM calculations, we attempted to investigate the proton transfer in enzyme catalysis associated with a few amino acid residues of motifs F and D.
| S-EPMC3531290 | biostudies-literature
Project description:Dengue virus (DENV) infects 50-100 million people worldwide per year, causing severe public health problems. DENV RNA-dependent RNA polymerase, an attractive target for drug development, catalyzes de novo replication of the viral genome in three phases: initiation, transition, and elongation. The aim of this work was to characterize the mechanism of nucleotide addition catalyzed by the polymerase domain of DENV serotype 2 during elongation using transient kinetic methods. We measured the kinetics of formation of the elongation complex containing the polymerase and a double-stranded RNA by preincubation experiments. The elongation complex assembly is slow, following a one-step binding mechanism with an association rate of 0.0016 ± 0.0001 μM(-1) s(-1) and a dissociation rate of 0.00020 ± 0.00005 s(-1) at 37 °C. The elongation complex assembly is 6 times slower at 30 °C and requires Mg(2+) during preincubation. The assembled elongation complex incorporates a correct nucleotide, GTP, to the primer with a K(d) of 275 ± 52 μM and k(pol) of 18 ± 1 s(-1). The fidelity of the polymerase is 1/34,000, 1/59,000, 1/135,000 for misincorporation of UTP, ATP, and CTP opposite CMP in the template, respectively. The fidelity of DENV polymerase is comparable with HIV reverse transcriptase and the poliovirus polymerase. This work reports the first description of presteady-state kinetics and fidelity for an RNA-dependent RNA polymerase from the Flaviviridae family.
| S-EPMC3023504 | biostudies-literature