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)

Purified RNA polymerase II (pol II) from the yeast Saccharomyces cerevisiae pauses without releasing at many locations during in vitro transcription. Pausing can be induced by intrinsic DNA sequence as well as by specific DNA bound proteins such as the RNA pol I termination factor, Reb1p, or lac repressor. Addition of rho termination factor from E. coli induces RNA pol II to release at all of these pause sites. Rho-induced release of pol II requires both a rho binding site in the transcript upstream of the pause sites as well as hydrolysis of ATP. In contrast, rho factor has no effect on either pausing or release by RNA pol I or III. When combined with previous observations, these results suggest that RNA pol II may terminate by a mechanism closely related to the rho-dependent mechanism of prokaryotes. In contrast, pol I and III appear to utilize a mechanism more related to the rho-independent terminators of prokaryotes.
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PMID:Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III. 956 Feb

The transcription factors NusA and NusG from Escherichia coli are modulators of the RNA polymerase elongation reaction and Rho-dependent transcription termination. NusA decreases the elongation rate and termination efficiency while NusG increases both activities. Both Nus factors are able to physically interact with Rho and with RNA polymerase. Experiments with purified components designed to determine whether these factors act independently or competitively showed that the change in elongation rate was a composite of their individual effects, that the combined effect on termination was dependent on the reaction conditions and that the two factors do not compete for their sites of action for either effect. The two factors were also found not to enhance significantly the slight (20%) inhibition of elongation caused by 200 microM guanosine 3',5'-bisdiphosphate (ppGpp) during transcription in vitro. The results also show that the effects of NusA and NusG on RNA polymerase elongation and Rho function are contrary to the inverse relationship between elongation and termination that is expected for a kinetic coupling of Rho action to RNA polymerase elongation. This property suggests that in addition to their known actions on RNA polymerase that influence the length of pausing, these factors act on some other rate-limiting step of the Rho-dependent termination process.
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PMID:Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli. 957 Oct 53

Escherichia coli transcription termination protein Rho, an RNA-dependent ATPase, disrupts transcription complexes, releasing RNA and allowing RNA polymerase to recycle. Homohexameric Rho binds three molecules of MgATP in a single class of catalytically competent sites. In rapid mix chemical quench experiments, when Rho saturated with ATP was mixed with RNA and the reaction was quenched after various times, hydrolysis of the three bound ATP molecules was not simultaneous. A hydrolysis burst of one molecule of ATP per hexamer occurred at >300 s-1, followed by steady-state hydrolysis at 30 s-1 per hexamer. The burst also shows that a step following ATP hydrolysis is rate-limiting for overall catalysis and requires communication among the three catalytic sites during net ATP hydrolysis. The rate of hydrolysis of radiolabeled ATP when one labeled and two unlabeled ATP molecules are bound indicates a sequential pattern of hydrolysis. Positive cooperativity of catalysis occurs among the catalytic sites of Rho; when only one ATP molecule is bound per hexamer, ATP hydrolysis upon addition of RNA is 30-fold slower than when ATP is saturating. These behaviors are comparable to those of F1-type ATPases, with which Rho shares a number of structural features.
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PMID:Sequential hydrolysis of ATP molecules bound in interacting catalytic sites of Escherichia coli transcription termination protein Rho. 975 83

There is a kinetic limitation to Rho function at the first intragenic terminator in the lacZ gene (tiZ1) which can be overcome by NusG: Rho can terminate transcription with slowly moving, but not rapidly moving, RNA polymerase unless NusG is also present. Here we report further studies with two other Rho-dependent terminators that are not kinetically limited (tiZ2 and lambda tR1) which show that the requirement for NusG depends on the properties of the terminator and its location in the transcription unit. NusG is also shown to increase the rate of Rho-mediated dissociation of transcription complexes arrested at a specific termination stop point in the tiZ1 region and the rates of dissociation with three different Rho factors and two different terminators correlated with their sensitivity to RNA polymerase elongation kinetics. These results suggest a model of NusG function which involves an alteration in the susceptibility of the transcription complex to Rho action which allows termination to occur within the short kinetic window when RNA polymerase is traversing the termination region.
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PMID:Activation of Rho-dependent transcription termination by NusG. Dependence on terminator location and acceleration of RNA release. 998 75

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo. We show that it is also essential for Nun termination at lambda nutL. Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR. The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL. The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.
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PMID:Escherichia coli nusG mutations that block transcription termination by coliphage HK022 Nun protein. 1020 50

A chemotaxis gene cluster from Treponema denticola (Td), a pathogenic spirochete associated with human periodontal diseases, was cloned, sequenced, and analyzed. The gene cluster contained three chemotaxis (che) genes (cheA, cheW, and cheY) and an open reading frame (cheX) that is homologous with Treponema pallidum (Tp) and Borrelia burgdorferi (Bb) cheX. The Td che genes have the same transcriptional orientation with a sigma 70-like promoter located upstream of cheA and a stem-loop structure characteristic of a Rho-independent transcriptional terminator downstream of cheY. Primer extension analysis identified a transcriptional start point six nucleotides (nt) downstream of the -10 (TAAAAA) promoter sequence. Reverse-transcriptase-polymerase chain reaction (RT-PCR) data indicated that cheA through cheY are co-transcribed and suggested that transcription is terminated after cheY. The gene organization of the Td che operon is identical to that of the Tp che operon. Southern blot analysis indicated the presence of one copy of each che gene on the Td genome. The cheA, cheW, cheX, and cheY genes are 2403, 1332, 462, and 438nt long, respectively, and encode proteins with predicted molecular masses of 88.2, 49.7, 16.8, and 16. 0kDa, respectively. Functional domains of the T. denticola CheA and CheY proteins are highly conserved with those of the Escherichia coli (Ec) CheA and CheY proteins. Phylogenetic analysis of Td CheY indicated that it is closely related to Tp CheY and Bb CheY3.
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PMID:Molecular characterization of a chemotaxis operon in the oral spirochete, Treponema denticola. 1033 22

To terminate transcription in E. coli, Rho protein binds an RNA loading site on the nascent transcript, translocates 5'--> 3' along the RNA in an ATP-driven process, and, upon reaching the transcription elongation complex, brings about RNA release. Thus, the Rho-dependent termination process can be viewed, in part, as a kinetic competition between the rate of transcript elongation by RNA polymerase (RNAP) and the rate of Rho translocation along the nascent transcript. In the context of this model, NusG, which is an essential E. coli protein, regulates Rho-dependent termination in an apparently paradoxical way, increasing the rate of transcription elongation of E. coli RNAP in the absence of Rho while also shifting the sites of Rho-dependent termination upstream on the template. Here we investigate the regulation of Rho-dependent termination by NusG. Analytical ultracentrifugation was used to establish the existence of a stable complex of NusG and Rho and to demonstrate a stoichiometry of one NusG monomer per Rho hexamer. Surface plasmon resonance was used to examine the kinetics of the formation and dissociation of the NusG-Rho complex, yielding an association rate constant (k(on)) of 2.8 (+/-0.8) x 10(5) M(-)(1) s(-)(1), a dissociation rate constant (k(off)) of 3.9 (+/-0.7) x 10(-)(3) s(-)(1), and a calculated equilibrium (dissociation) constant (K(d)) of 1.5 (+/-0.3) x 10(-)(8) M. The apparent stability of the NusG-Rho complex is insensitive to changes in salt (potassium acetate) concentration between 0.05 and 0.15 M. The translocation and transcription termination activities of Rho at saturating NusG concentrations were, however, both sensitive to salt concentration over this range, suggesting that these activities do not directly reflect the stability of the NusG-Rho complex. Rho-dependent termination could be demonstrated for transcription complexes in which E. coli RNAP had been substituted by either bacteriophage SP6 or T7 RNAP. NusG, however, was not active in transcription termination assays with either of these phage RNAPs. Thus, we conclude that NusG modulates Rho-dependent termination by interacting specifically with the RNAP of the E. coli elongation complex to render the complex more susceptible to the termination activity of Rho.
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PMID:Regulation of rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex. 1082 31

Mutations conferring resistance to the antibiotic rifampicin (Rif(r)) occur at specific sites within the ss subunit of the prokaryotic RNA polymerase. Rif(r) mutants of Escherichia coli are frequently altered in the elongation and termination of transcription. Rif(r) rpoB mutations were isolated in Bacillus subtilis and their effects on transcription elongation factor NusG and Rho-dependent termination were investigated. RNase protection assay, Northern analysis and the expression of nusG-lacZ fusions in cells with an inducible NusG suggested the B. subtilis nusG gene was autoregulated at the level of transcription. Rif(r) mutations that changed residue Q469 to a basic residue (Q469K and Q469R) enhanced autoregulation of nusG. A mutant expressing a truncated form of NusG, due to a nonsense mutation within the nusG gene, was isolated on the basis of the loss of autoregulation. The mechanism of autoregulation was found to be independent both of transcription termination factor Rho and of the promoter transcribing nusG. Autoregulation required sequences within the 5' coding sequence of the nusG gene or immediately upstream. This is the first evidence from any bacterium that Rif(r) RNA polymerases can display altered transcription regulation by NusG.
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PMID:Mutations in the ss subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG. 1110 62

Escherichia coli ribosomal RNA (rRNA) operons contain antitermination motifs necessary for forming terminator-resistant transcription complexes. In preliminary work, we isolated 'antiterminating' transcription complexes and identified four new proteins potentially involved in rRNA transcription antitermination: ribosomal (r-) proteins S4, L3, L4 and L13. We show here that these r-proteins and Nus factors lead to an 11-fold increase in terminator read-through in in vitro transcription reactions. A significant portion of the effect was a result of r-protein S4. We show that S4 acted as a general antitermination factor, with properties very similar to NusA. It retarded termination and increased read-through at Rho-dependent terminators, even in the absence of the rRNA antiterminator motif. High concentrations of NusG showed reduced antitermination by S4. Like rrn antitermination, S4 selectively antiterminated at Rho-dependent terminators. Lastly, S4 tightly bound RNA polymerase in vivo. Our results suggest that, like NusA, S4 is a general transcription antitermination factor that associates with RNA polymerase during normal transcription and is also involved in rRNA operon antitermination. A model for key r-proteins playing a regulatory role in rRNA synthesis is presented.
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PMID:Ribosomal protein S4 is a transcription factor with properties remarkably similar to NusA, a protein involved in both non-ribosomal and ribosomal RNA antitermination. 1144 22

Interactions between the antiterminator NusB and boxA elements in the nut sites are necessary to ensure lambda N-mediated processive antitermination. Similarly, in the bacterial cell, interactions between NusB and boxA elements help RNA polymerase to counteract polarity during transcription of rrn operons. We analyzed the effects of NusB on intragenic termination at the level of two tandem terminators located in the hisG cistron, GTTE1 and GTTE2. Unexpectedly, we found that NusB enhances transcription termination at the sub-optimal Rho site GTTE1. Moreover, site-directed mutagenesis of a boxA homolog located within GTTE1 and the masking of this element by translating ribosomes demonstrated that the recruitment of NusB in the termination complex is mediated by a boxA element. The mutated boxA also abolishes the formation of a NusB-dependent complex on GTTE1 RNA. On the whole, results provide evidence that interactions between NusB and boxA can enhance Rho-dependent termination.
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PMID:The antiterminator NusB enhances termination at a sub-optimal Rho site. 1149 Dec 88


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