ArticleThe Journal of biological chemistry2012
Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex.
Article in The Journal of biological chemistry, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed.
- Slow PPThe Journal of biological chemistry · 2026Article
- Optimization of a malachite green-pyrophosphatase assay for the Zika virus RNA-dependent RNA polymerase and preliminary identification of natural product modulators.Frontiers in drug discovery · 2026Article
- Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.Nucleic acids research · 2023Article
- Structural basis of transition from initiation to elongation in de novo viral RNA-dependent RNA polymerases.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- An induced-fit de novo initiation mechanism suggested by a pestivirus RNA-dependent RNA polymerase.Nucleic acids research · 2021Article
- Article
- Hepatitis C Viral Replication Complex.Viruses · 2021Review
- Crystal structure of a tick-borne flavivirus RNA-dependent RNA polymerase suggests a host adaptation hotspot in RNA viruses.Nucleic acids research · 2021Article
- Rate-limiting pyrophosphate release by hepatitis C virus polymerase NS5B improves fidelity.The Journal of biological chemistry · 2020Article
- Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis.Nature communications · 2020Article
- Zinc(II)-The Overlooked Éminence Grise of Chloroquine's Fight against COVID-19?Pharmaceuticals (Basel, Switzerland) · 2020Review
- Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs.The Journal of biological chemistry · 2020Article
- Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase.bioRxiv : the preprint server for biology · 2020Article
- A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase.PLoS pathogens · 2020Article
- Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells.Viruses · 2019Article
- A unique intra-molecular fidelity-modulating mechanism identified in a viral RNA-dependent RNA polymerase.Nucleic acids research · 2018Article
- NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B.The Journal of biological chemistry · 2017Article
- The Battle of RNA Synthesis: Virus versus Host.Viruses · 2017Review
- Cystoviral RNA-directed RNA polymerases: Regulation of RNA synthesis on multiple time and length scales.Virus research · 2017Review
- Picornaviral polymerase structure, function, and fidelity modulation.Virus research · 2017Review
Corrections and comments
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Authors and funding
5 authors.
Funding
Abstract
NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.