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At least 37 records · Page 2Linked to original sources
PubMed1977

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toIC and dnaG, at 66 min.

Chromosome Mapping↗
PubMed1977

Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster.

RNA polymerase (RNA nucleotidyltransferase) B (or II) and histone H1 of Drosophila melanogster were localized on salivary gland polytene chromosomes using the indirect immunofluorescence technique. RNA polymerase B is present almost exclusively in puffs and interband regions, whereas histone H1 is found primarily in bands. The puff at region 3C, known to be transcriptionally active in larval salivary glands, gives a bright fluorescence with antibodies against RNA polymerase B. This fluorescence disappears after exposure of the larvae to 37 degrees for 45 min. The heat shock treatment results in a general reduction of fluorescence intensity with the appearance of brightly staining heat shock puffs. Heat-induced removal of RNA polymerase molecules from a puff does not immediately alter its morphology. We propose than an interband represents that fraction of the total number of gene copies in a band that are active, the inactive copies being present in a condensed form in the adjacent band. Large puffs would originate through the decondensation and activation of most or all gene copies in a band.

Animals↗
PubMed1979

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.

Cell-Free System↗
PubMed2026

Slow PPi release enhances fidelity of the SARS-CoV-2 RNA dependent RNA polymerase.

Viral RNA-dependent RNA polymerases (RdRps) must balance replication speed with fidelity, preserving genome integrity while permitting enough variability for viral adaptation. The SARS-CoV-2 RdRp complex (non-structural protein 12/7/8) achieves this through the interplay of its intrinsic replication fidelity and a potential proofreading exonuclease complex (NSP10/14). Here, we comprehensively quantify the intrinsic fidelity of the SARS-CoV-2 RdRp through direct pre-steady-state kinetic analyses of nucleotide incorporation across all possible templating bases paired with incoming nucleotides. We discovered substantial variation in discrimination against mismatches ranging from one error in 103 to 108 (median of 105). Crucially, our data reveal a slow pyrophosphate release step that significantly enhances fidelity by effectively introducing a kinetic checkpoint after nucleotide incorporation. The error rates we measured for the RdRp align closely with observed in vivo mutation rates, suggesting that the exonuclease complex may play a less critical role than previously assumed in correcting mistakes during polymerization. These insights advance our understanding of SARS-CoV-2 replication fidelity, and the role of various subcomplexes in genome maintenance and adaptation.

SARS-CoV-2↗
PubMed1977

Selective and accurate transcription of the Xenopus laevis 5S RNA genes in isolated chromatin by purified RNA polymerase III.

Chromatin isolated from immature oocytes was found to contain an endogenous RNA polymerase activity (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) that synthesizes predominately 5S RNA. However, the levels of total RNA synthesis and 5S RNA synthesis in chromatin were each stimulated 10- to 50-fold by an exogenous RNA polymerase III purified from X. laevis oocytes. The 5S genes in chromatin were transcribed by the exogenous enzyme in a highly selective (3000-fold above random) and predominately asymmetric fashion. A significant fraction of 5S RNA sequences were also found in a discrete transcript, approximately 5S in size. Total RNA synthesis was significantly stimulated when chromatin was transcribed by oocyte RNA polymerase I, murine RNA polymerase II, and low levels of Escherichia coli RNA polymerase. However, these enzymes did not significantly stimulate 5S RNA synthesis above the endogenous levels. Both homologous oocyte RNA polymerase I and III and E. coli RNA polymerase transcribed the 5S genes in deproteinized DNA to approximately the same extent (severalfold above random) and both the sense and anti-sense strands of the gene were transcribed. It appears, therefore, that both chromatin-associated components and a purified RNA polymerase III are necessary and sufficient for the selective and accurate transcription of the 5S RNA genes in vitro.

Animals↗
PubMed1975

Uukuniemi virus contains an RNA polymerase.

An RNA-dependent RNA polymerase activity has been found associated with Uukuniemi virions. The enzyme activity is expressed only after disrupting the virions with the nonionic detergent Triton X-100 and is absolutely dependent on Mn2+, whereas Mg2+ is not required, a finding that distinguishes this polymerase from those of other enveloped minus-strand RNA viruses. Within the range pH 7.2 to 8.5 no distinct optimum was found. The optimum temperature was between 37 and 40 C. The reaction was not inhibited by actinomycin D, rifampin, or DNase, whereas RNase was completely inhibitory. The partially RNase-resistant product consisted of rather small-sized RNA, which contained sequences complementary to Uukuniemi virus RNA as shown by hybridization to the template L, M, and S RNA species of Uukuniemi virus.

Arboviruses↗
PubMed1977

Transcription of double-stranded RNA by Escherichia coli DNA-dependent RNA polymerase.

Double-stranded RNA of some virus genomes can be used as template for the DNA-dependent RNA polymerase purified from Escherichia coli. The RNA synthesis requires all four nucleoside triphosphates and manganese ions and is dependent on the presence of sigma subunit. The reaction is inhibited by rifampicin, streptolydigin and ethidium bromide, but not by DNase and actinomycin D which does not bind to double-stranded RNA. The template activity of double-stranded RNA from various viruses is different in each case. The order of template efficiency is Penicillum chrysogenum virus greater than cytoplasmic polyhedrosis virus greater than rice dwarf virus greater than reovirus. The product obtained using cytoplasmic polyhedrosis virus double-stranded RNA as template is single-stranded and hybridizes specifically to the denatured template RNA. One of the major 5'-starting nucleotide sequences of the product RNA is pppA-A-Y--. These results indicate that transcription in vitro of double-stranded RNA by E. Coli RNA polymerase is initiated at specific sites on the template.

Animals↗