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RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.


Papers
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Journal ArticleDOI
TL;DR: In this article, the authors describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions.
Abstract: In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

294 citations

Journal ArticleDOI
TL;DR: In this paper, the authors used a two-dimensional gel analysis to identify 96 sites of very high DNA polymerase binding in wild-type cells, including open reading frames (ORFs) of highly transcribed RNA polymerase II genes.

294 citations

Journal ArticleDOI
TL;DR: A comprehensive assessment of the distribution of H-NS within the E. coli genome is presented, light is shed on the mechanism underlying the transcriptional regulation by H- NS, and new insight into bacterial genome evolution is provided.
Abstract: Heat-stable nucleoid-structuring protein (H-NS) is one of the main nucleoid proteins expressed in exponentially growing Escherichia coli cells. In addition to a role in nucleoid organization, H-NS functions as a pleiotropic regulator of gene expression. The genome-wide distribution of H-NS, compared with the distribution of RNA polymerase and transcriptionally active genes, was investigated using a high-density oligonucleotide chip. The new approach utilized in this study revealed that H-NS binds specifically to approximately 250 loci, covering >1000 genes, to maintain transcriptional inactivation. RNA polymerase was detected in >65% of H-NS binding sites with low or no transcriptional activity, indicating that the association of RNA polymerase to promoter regions is a general mode of transcription repression by H-NS. This study also revealed that most H-NS bound DNA have been horizontally acquired, which indicates that repression of inappropriate gene expression by H-NS plays an important role in the diversification of the E. coli genome. This study presents a comprehensive assessment of the distribution of H-NS within the E. coli genome, sheds light on the mechanism underlying the transcriptional regulation by H-NS, and provides new insight into bacterial genome evolution.

294 citations

Journal ArticleDOI
TL;DR: Plants transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1 were resistant to infection and accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.
Abstract: Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.

293 citations

Journal ArticleDOI
TL;DR: Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B.
Abstract: Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

292 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202358
2022201
2021222
2020200
2019116
2018118