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)

The N gene protein of bacteriophage lambda prevents termination of transcription by E. coli RNA polymerase. We describe here the conditions of a cell-free reaction system in which pure N stimulates net transcription up to tenfold and therefore nearly stoichiometrically modifies transcribing RNA polymerase molecules. The reaction contains micrococcal nuclease-treated S100 extract derived from E. coli and a plasmid template DNA containing the lambda early promoter PL, the N utilization site nutL, and the Rho-dependent terminator tL1. Stimulation by N in this system is specific and biologically relevant since it is absent with vector pBR322 DNA and with extracts derived from E. coli strains bearing the nusA1 and nusE71 mutations known to block N function in vivo. We use the system to provide further evidence that ribosomes are not necessary for N function and to demonstrate the direct involvement in N function of the NusA protein of E. coli.
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PMID:Efficient modification of E. coli RNA polymerase in vitro by the N gene transcription antitermination protein of bacteriophage lambda. 315 83

Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay. Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA. RNA molecules were preferentially released from enzyme molecules located within the termination region. Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor. Pretreatment of complexes with ribonuclease prevented dissociation of DNA. Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor. In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter. Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.
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PMID:Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase. 388 62

Transcription of T7 DNA by purified Escherichia coli RNA polymerase without added factors produces long RNA molecules that begin near the left end of T7 DNA and terminate at the end of the early region. An endonuclease has been isolated from uninfected E. coli that cleaves these long RNAs at five specific sites to generate RNA molecules essentially the same as the early T7 RNAs observed in vivo. This sizing factor, which may be RNase III, can act during or after RNA synthesis. Synthesis of early RNA chains has been shown to start at three strong initiators, spaced about 150-200 base-pairs apart near the left end of T7 DNA. Thus, the five cleavages by sizing factor generate the five early messenger RNAs of T7 plus three overlapping RNAs from the promoter region. RNA chains that are started at two of the strong initiators begin with A; those started at the third begin with G. A few minor initiators have also been observed, from which only short chains seem to be synthesized. Their locations in T7 DNA have not been mapped. Rho factor does not appear to be needed to generate any of these early T7 RNAs.
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PMID:T7 early RNAs are generated by site-specific cleavages. 457 24

We have conducted a detailed investigation of in vitro transcription from the bacteriophage lambda PR promoter in order to examine various aspects of the mechanism of rho-dependent termination. In these studies, we have focused particularly on nucleotide sequence specificity, both at the termini and at potential rho-binding sites on the mRNA, and on the relationships between elongation, pausing, and termination. Rho-terminated transcripts from restriction fragment templates have been analyzed by polyacrylamide gel electrophoresis, and termination efficiencies have been established by densitometry of autoradiographs. Termination sites on the template have been located by comparing the electrophoretic mobilities of terminated transcripts with those of transcripts of known length that have been artificially terminated by the incorporation of 3'-O-methyl nucleotides. We have identified five discrete rho-dependent termination sites located between 290 and 450 base pairs downstream from the lambda PR promoter. These rho-dependent 3'-termini are somewhat heterogeneous in details of sequence and potential RNA secondary structure, but all possess features that appear to be characteristic of RNA polymerase elongation pausing sites (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9565-9574). The efficiency of termination at individual sites ranges from 20 to 70% under the usual in vitro transcription conditions; termination is inhibited by increasing the monovalent salt concentration. Lowering nucleoside triphosphate substrate concentrations increases termination efficiency at some sites located 290 or more base pairs downstream from PR, but does not enhance termination at sites closer to PR. The substitution of inosine for guanosine residues in the transcript, which decreases the stability of the RNA-DNA hybrid and of secondary structure in the nascent mRNA, results in strong rho-dependent termination at several new sites located 100 to 260 base pairs downstream from PR. In Morgan et al. (cited above), data on RNA polymerase elongation pausing as a function of reaction conditions are correlated with these termination results, and a general model for rho-dependent termination is discussed.
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PMID:Rho-dependent termination of transcription. I. Identification and characterization of termination sites for transcription from the bacteriophage lambda PR promoter. 622 29

The Escherichia coli protein NusG is known to modulate Rho-dependent transcription termination in vivo. We have shown that it can also alter the pattern of Rho-dependent RNA endpoints in vitro, at lower NusG concentrations than can be explained by reported interactions between NusG and Rho or RNA polymerase. Three observations in vitro now suggest a model to account for these effects of NusG on Rho-dependent termination. First, the presence of NusG circumvents the interference with Rho function caused by adding DNA oligonucleotides complementary to particular segments of the Rho binding site. Second, when NusG is added to stalled elongation complexes, the off-rate of Rho from nascent RNA is slowed. Third, NusG associates stably with the elongation complex only when Rho is also present and bound to the nascent RNA. Our observations are consistent with a model in which NusG and Rho participate in an interdependent association with the transcribing RNA polymerase and the nascent RNA to facilitate the recognition and use of termination signals. Common structural and functional features shared with complexes that carry out processive antitermination are discussed.
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PMID:A quaternary transcription termination complex. Reciprocal stabilization by Rho factor and NusG protein. 752 72

The boxA sequences of the E. coli ribosomal RNA (rrn) operons are sufficient to cause RNA polymerase to read through Rho-dependent transcriptional terminators. We show that a complex of the transcription antitermination factors NusB and ribosomal protein S10 interacts specifically with boxA RNA. Neither NusB nor S10 binds boxA RNA on its own, and neither NusA nor NusG affects the interaction of the NusB-S10 complex with boxA RNA. Mutations in boxA that impair its antitermination activity compromise its interaction with NusB and S10, suggesting that ribosomal protein S10 regulates the synthesis of ribosomal RNA in bacteria. RNA containing the closely related boxA sequence from the bacteriophage lambda nutR site is not stably bound by NusB and S10. This probably explains why antitermination in phage lambda depends on the phage lambda N protein and the boxB component of the nut site, in addition to boxA.
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PMID:Recognition of boxA antiterminator RNA by the E. coli antitermination factors NusB and ribosomal protein S10. 767 81

Rho-dependent transcription termination at certain terminators in Escherichia coli also depends on the presence of NusG [Sullivan, S. L. & Gottesman, M. E. (1992) Cell 68, 989-994]. We have found that termination at the first intragenic terminator in lacZ (tiZ1) is strongly dependent on NusG when transcription is done in vitro with the concentrations of NTPs found in vivo. With a lower level of NTPs, and consequently a slower rate of RNA-chain growth, Rho causes some termination by itself that is enhanced with NusG. These results suggest that NusG serves to overcome a kinetic limitation of Rho to function at certain terminators. At a second intragenic terminator within the lacZ reading frame (tiZ2) the efficiency of Rho-mediated termination was unaffected by either NusG or by RNA polymerase elongation kinetics. Thus, using purified components and intracellular levels of NTPs, we have confirmed the in vivo finding that certain Rho-dependent terminators also depend on NusG, whereas others do not.
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PMID:NusG is required to overcome a kinetic limitation to Rho function at an intragenic terminator. 776 93

Escherichia coli Rho factor is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine-containing RNA target sites, subsequent RNA-dependent ATP hydrolysis, and an RNA-DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revelations.
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PMID:Rho and RNA: models for recognition and response. 802 88

NusA and Rho are essential Escherichia coli proteins that influence transcription elongation and termination. We show that an E. coli derivative unable to express NusA, because its sole nusA gene contains a large deletion/substitution, is viable providing that the bacterium also carries a rho mutation that reduces transcription termination. This Rho-mediated suppression is not allele specific, since either a mutation changing amino acid 134 [rho(E134D)] or a mutation changing amino acid 352 (rho1) allows growth of a nusA-deleted E. coli. However, both rho mutations similarly decrease transcription termination 8- to 9-fold. We propose that the essential role of NusA is to enhance pausing of RNA polymerase at certain sites, permitting tight coupling of transcription and translation. This coupling interferes with Rho access to and/or movement on the nascent RNA and blocks premature termination of transcription. Thus, NusA-dependent coupling should be less important in a mutant with low Rho activity. The fact that E. coli grows without NusA argues that NusA should be considered an accessory factor rather than a subunit of RNA polymerase.
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PMID:Reduced Rho-dependent transcription termination permits NusA-independent growth of Escherichia coli. 805 17

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.
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PMID:Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis. 805 27


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