Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin.
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PMID:Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases. 768 33

A stable ternary transcription complex was formed with either wheat germ or yeast RNA polymerase II using a ribotrinucleotide primer (GpCpG) to initiate transcription on a short synthetic single-strand DNA template. The template was designed to limit the incorporation of a photoprobe S4-UMP (4-thio-UMP) to a unique position at the 3' terminus of the transcript. The resulting stable ternary transcription complex was photolyzed to cross-link the bound transcript ([32P]-labeled by the incorporation of [alpha-32P]CMP) with the protein domain at or near the active site. Separation of the protein components by electrophoresis in polyacrylamide gel containing SDS and analysis by autoradiography and silver staining revealed that for either enzyme only the largest subunit was [32P] labeled.
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PMID:Wheat germ and yeast RNA polymerase II: photoaffinity labeling by 4-thiouracil 5'-monophosphate positioned uniquely at the 3' end of an enzyme-bound [32P]-containing transcript. 844 67

We studied capsule-defective (Cap-) serogroup B meningococcal mutants created through Tn916 or omega-fragment mutagenesis. The Cap- phenotypes were the results of insertions in three of four linked genes (synX, synC, and synD) involved in CMP-N-acetylneuraminic acid and polysialic acid capsule biosynthesis, and in ctrA the first of four linked genes involved in capsule membrane transport. Mutations in the CMP-N-acetylneuraminic acid biosynthesis genes synX and synC caused defects in lipooligosaccharide sialylation but not mutations in the putative (alpha2 -> 8)-linked polysialyltransferase (synD) or in ctrA. Reverse transcriptase PCR studies indicated that the four biosynthesis genes (synX to -D) and the capsule transport genes (ctr to -D) were separately transcribed as operons. The operons were separated by a 134-bp intergenic region. Primer extension of synX and ctrA demonstrated that transcription of the operons was divergently initiated from adjacent start sites present in the intergenic region. Both transcriptional start sites were preceded by a perfect -10 Pribnow promoter binding region. The synX to -D, but not the ctrA to -D, transcriptional start site was preceded by a sequence bearing strong homology to the consensus sigma 70 -35 promoter binding sequence. Both promoters showed transcriptional activity when cloned behind a lacZ reporter gene in Escherichia coli. Our results confirm the intrinsic relationship between polysialic acid capsule biosynthesis and lipooligosaccharide sialylation pathways in group B Neisseria meningitidis. Our study also suggests that the intergenic region separating the synX to -D and ctrA to -D operons is an important control point for the regulation of group B capsule expression through coordinated transcriptional regulation of the synX to -D and drA to -D promoters.
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PMID:Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region. 876 31

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
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PMID:Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression. 895 11

Vaccinia virus RNA polymerase terminates transcription downstream of a UUUUUNU signal in the nascent RNA. Transduction of the RNA signal to the elongating polymerase requires a termination factor (vaccinia termination factor/capping enzyme) and is coupled to the hydrolysis of ATP. It was shown previously that incorporation of 5-bromouracil or 5-iodouracil within the UUUUUNU element abolishes termination by preventing factor-dependent release of the nascent chain from the polymerase elongation complex. Here, we report that termination is prevented by phosphorothioate substitution at UMP residues in the nascent RNA. In contrast, phosphorothioate substitution at AMP, CMP, and GMP nucleotides does not inhibit termination. Thus, the action of a eukaryotic termination factor entails recognition of the nucleotide bases and the phosphate groups of the target sequence in nascent RNA.
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PMID:Transcription termination by vaccinia RNA polymerase entails recognition of specific phosphates in the nascent RNA. 899 14

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.
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PMID:Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6. 1067 13

Ribavirin is administered in combination with interferon-alpha for treatment of hepatitis C virus (HCV) infection. Recently, we demonstrated that the antiviral activity of ribavirin can result from the ability of a viral RNA polymerase to utilize ribavirin triphosphate and to incorporate this nucleotide with reduced specificity, thereby mutagenizing the genome and decreasing the yield of infectious virus (Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379). In this study, we performed a quantitative analysis of a novel HCV RNA polymerase derivative that is capable of utilizing stably annealed primer-template substrates and exploited this derivative to evaluate whether lethal mutagenesis of the HCV genome is a possible mechanism for the anti-HCV activity of ribavirin. These studies demonstrate HCV RNA polymerase-catalyzed incorporation of ribavirin opposite cytidine and uridine. In addition, we demonstrate that templates containing ribavirin support CMP and UMP incorporation with equivalent efficiency. Surprisingly, templates containing ribavirin can also cause a significant block to RNA elongation. Together, these data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase. We discuss the implications of this work on the development of nucleoside analogs for treatment of HCV infection.
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PMID:Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. 1160 68

Cell-free synthesis of citrus exocortis viroid (CEV) in nuclei-rich preparations from infected Gynura aurantiaca was optimum at 18-24 degrees C. Incubation of reaction mixtures at higher temperatures (30-36 degrees C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the Mg(2+) optimum (2.5-5 mM) for CEV synthesis was lower than that for total [(32)P]CMP incorporation (10 mM), the response to Mn(2+) ion was distinctly different. Whereas maximum total activity was observed in 1 mM Mn(2+) with a pronounced reduction (80%) in 5 mM Mn(2+), CEV synthesis was maintained in 1-15 mM Mn(2+). Inhibition of alpha-amanitin-sensitive CEV synthesis in 200 mM (NH(4))(2)SO(4) resembles the reaction of RNA polymerase II on a free nucleic acid template. However, detection of trace levels of alpha-amanitin-resistant CEV synthesis activity inhibited by low (NH(4))(2)SO(4) concentrations (25 mM) suggests the possible involvement of RNA polymerase I- and/or III-like activity.
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PMID:Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity. 1659 89

Substrate specificities of three viral replicative polymerases of different origins (HIV reverse transcriptase, hepatitis C virus RNA polymerase, and herpes virus DNA polymerase) towards 2'F-NTP were studied. Activated DNA, polyA-oligoUs and (2'F-A)20-oligoU6-complexes were used as templates. It was shown that all DNA polymerases studied can incorporate 2'F-NMP into the 3'-end of primer-template complexes. HIV reverse transcriptase and herpes virus DNA polymerase can elongate synthesis with both dNTP and 2'F-NTP. Homopolymer (2'F-A)20 can serve as a template for polymerization of both UTP and 2'F-UTP,-catalyzed by hepatitis C virus polymerase although with efficacy about 5 to 10-fold lower in comparison with natural primertemplate complex. Pyrophosphorolysis reaction of 2'F-CMP residue at 3'-end of primer catalyzed with HIV reverse transcriptase is going by two orders of magnitude less effective if compared with natural dNMP residue at the same system.
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PMID:[2'fluoro derivatives of nucleosides as substrates of viral replicative nucleotide polymerases]. 2584 69


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