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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)

Bacillus subtilis RNA polymerase holoenzyme consists of the subunits beta', beta, sigma, alpha, delta, and omega. In sporulating bacteria and in bacteria infected with phages SP01 and SP82, this enzyme undergoes changes in subunit composition and transcriptional specificity that could play a regulatory role in gene transcription. Sporulating bacteria may contain a specific component that inhibits the activity of the sigma subunit of polymerase probably by interfering with the binding of sigma-polypeptide to core enzyme. The hypothetical inhibitor may be metabolically unstable, since its activity is rapidly depleted from sporulating cells in the presence of chloramphenicol. Inhibition of sigma-polypeptide activity may restrict the transcription of phage DNA an infected sporulating cells. Although lacking the sigma-subunit, RNA polymerase purified from sporulating cells contains sporulation-specific subunits of 85,000 and 27,000 daltons. In SP01-infected bacteria, the sigma-subunit is replaced by phage-induced subunits. Purified enzyme containing the protein product of SP01 regulatory gene 28 directs the transcription of phage middle genes in vitro, while enzyme containing phage-induced polypeptides V and VI preferentially copies late genes. Accurate transcription of middle and late genes in vitro requires the host delta-subunit of polymerase (or high ionic strength) but not sigma-subunit. Phage PBS2 induces an entirely new multisubunit RNA polymerase that specifically transcribes PBS2 DNA in vitro. This enzyme is synthesized de novo after infection and does not arise by modification of the B. subtilis holoenzyme.
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PMID:Bacillus subtilis RNA polymerase and its modification in sporulating and phage-infected bacteria. 5 49

The synthesis of 69 phage-specific polypeptides during the infection of Bacillus subtilis with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an Escherichia coli cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected B. subtilis and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides.
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PMID:Translation of RNAs synthesized in vivo and in vitro from bacteriophage SP82 DNA. 41 51

DNA-dependent RNA polymerase from Bacillus subtilis contains a 21,000 molecular weight (21K) peptide subunit. This subunit was purified and added to 21K-depleted polymerase isolated from both uninfected and SP85-infected B. subtilis. The effect of the subunit on total RNA synthesis, on enzyme-DNA binding, on RNA chain initiation and elongation, and on enzymatic specificity were examined. A comparison was made of the effects of the 21K peptide and NaCl on polymerase activity, RNA chain elongation, and symmetry of transcription of SP82 DNA. The addition of the 21K peptide to enzyme preparations lacking this subunit stimulated total polymerase activity 20 to 40%. In contrast, addition of NaCl at concentrations greater than 0.1 M significantly reduced polymerase activity. The 21K peptide appeared to alter the general affinity of the polymerase for DNA. The rate of RNA chain initiation was not affected by the 21K peptide, but RNA chain elongation was stimulated. Both the 21K peptide and NaCl increased the asymmetry of transcription of SP82 DNA by phage-modified polymerase. The 21K effect was related to the stimulation of elongation while high concentrations of NCl appeared to act at RNA chain initiation. RNAs synthesized in vitro by polymerase lacking and supplemented with the 21K peptide were translated by a Escherichia coli cell-free system. The 21K peptide had little direct effect on the selection of promoters in vitro as measured by this technique, but it dramatically increased the translatability of the product.
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PMID:Role of the 21,000 molecular weight polypeptide of Bacillus subtilis RNA polymerase in RNA synthesis. 41 17

Phage-specific RNA synthesized early in the infection of Bacillus subtilis with SP82 hybridizes to both heavy (H) and light (L) strands of SP82 DNA nearly equally. Phage RNA synthesized during the middle of the infection hybridizes preferentially to the H strand. The ratio of H/L strand binding of RNAs synthesized in vitro by RNA polymerases isolated from uninfected and infected cells resembles the ratios of early and middle phage RNA classes, respectively. This supports the conclusion that a modified RNA polymerase is required for the transcription of middle RNA classes.
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PMID:DNA strand specificity of transcripts produced in vivo and in vitro by RNA polymerase from SP82-infected Bacillus subtilis. 80 2

The Alu156 promoter isolated from the Bacillus subtilis bacteriophage SP82 is dependent on curved DNA upstream of the -35 region for efficient function. Short DNA insertions of 6-29 base pairs were used to simultaneously change the linear placement and rotational orientation of this curved DNA relative to the -35 region. When these mutant promoters were analyzed in vivo using transcriptional fusions with a chloramphenicol acetyltransferase gene, changes in the rotational orientation of the curved DNA correlated with changes in promoter function. The most efficient mutant promoters contained insertions of 11 and 21 base pairs, and insertions of 15 and 25 base pairs resulted in the least efficient mutant promoters. The importance of the proper rotational alignment of the curved DNA to promoter activity was also observed in vitro at the level of transcription of RNA polymerase binding. Based on the electrophoretic mobilities of DNA fragments containing the various insertion mutant promoters, there was a second region of curved DNA downstream of the insertion point. The findings are consistent with the idea that the curved DNA deflects the helix back toward the promoter-bound RNA polymerase molecule to allow the enzyme to interact directly with upstream DNA. These interactions are proposed to structure the DNA for the formation of the open promoter complex.
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PMID:Rotational orientation of upstream curved DNA affects promoter function in Bacillus subtilis. 254 69

The Bacillus subtilis RNA polymerase sigma 43 subunit and the phage SP82 encoded 28-kDa peptide are responsible for the binding of RNA polymerase to early and middle SP82 promoters, respectively. The delta peptide enhances the specificity of the interaction of B. subtilis RNA polymerase with these promoters. We have used sedimentation experiments to determine the effect of each of the three specificity factors, delta, sigma, and the 28-kDa peptide, on the binding of the other two factors to RNA polymerase core and the effect of NaCl on these binding equilibria. We show that sigma 43 and the 28-kDa peptide can each bind to RNA polymerase core at the same time as delta. Sigma 43 and the 28-kDa peptide have similar affinities to core at 0.1 M NaCl, but the 28-kDa peptide binds to core-delta more strongly than sigma 43. The implications of these findings with respect to the replacement of sigma 43 by the 28-kDa peptide and the mechanism of promoter search by B. subtilis RNA polymerase are discussed.
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PMID:Interactions of Bacillus subtilis RNA polymerase with subunits determining the specificity of initiation. Sigma and delta peptides can bind simultaneously to core. 309 10

The effect of DNA upstream of the -35 region on promoter function was examined using two promoters isolated from the Bacillus subtilis bacteriophage SP82. The affinity of RNA polymerase for the two promoters in vitro differed significantly. For each promoter the nucleotide sequence of the upstream DNA was characterized by the presence of successive runs of adenines with a 10-11-base pair periodicity. DNA fragments with the polyadenine-containing upstream DNA displayed aberrant electrophoretic mobilities when analyzed on polyacrylamide gels indicative of curved DNA. A series of mutant promoters in which the upstream DNA was deleted or altered was constructed. The curved DNA upstream of the -35 region was required for efficient RNA polymerase binding. Decreased in vitro transcription observed when the upstream DNA was deleted could be partially restored if the template was negatively supercoiled. Measurements of chloramphenicol acetyltransferase specific activity from B. subtilis strains carrying transcriptional fusions indicate that the curved upstream DNA stimulated transcription from the promoter with the weaker affinity for RNA polymerase. The curved DNA reduced the in vivo activity of the promoter with the strong affinity for RNA polymerase. One function of the curved upstream DNA may be to provide RNA polymerase-promoter interactions that facilitate open complex formation.
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PMID:Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis. 313 65

the ability of the core isolated from Escherichia coli RNA polymerase to interact with specificity-determining subunits isolated from Bacillus subtilis RNA polymerase has been determined by measuring the transcription of "early" and "middle" genes of phage SP82. Two specificity-determining subunits were tested: the sigma subunit and a 28,000 dalton (28 K) peptide isolated from a modified polymerase produced at approximately 8 min after infection of B. subtilis with SP82. Earlier experiments (Spiegelman, G. B. and Whiteley, H. R. (1978) Biochem. Biophys. Res. Commun. 81, 1058-1065) demonstrated that sigma and the 28K peptide are required for the recognition of early and middle gene promoters, respectively, by the B. subtilis core assembly. The present investigation showed that E. coli core interacted more efficiently with the B. subtilis sigma than with the 28K peptide, as judged by the rate of RNA synthesis. Early RNA was produced by the E. coli and B. subtilis holoenzymes and by E. coli core supplenented with B. subtilis sigma and only minor differences were found in comparisons of transcripts by hybridization and by electrophoretic analysis. Measurements of template specificity, the formation of stable enzyme . DNA complexes, and the hybridization of transcripts to fragments of SP82 DNA produced by digestion with restriction endonuclease Hha indicated that E. coli core supplemented with the 28K-supplemented E. coli core with those synthesized by the modified polymerase extracted from B. subtilis 8 min after infection with SP82 suggest that both preparations recognized the same initiation and termination sequences.
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PMID:The interaction of Escherichia coli core RNA polymerase with specificity-determining subunits derived from unmodified and SP82-modified Bacillus subtilis RNA polymerase. 616 Jan 56

A restriction map was constructed for the 17.5-kilobase SalI C fragment of SP82 DNA. Unfractionated SP82 DNA, the SalI C fragment, and restriction fragments derived from SalI-C were used as templates for in vitro synthesis by the Bacillus subtilis RNA polymerase, and the resulting transcripts were analyzed by gel electrophoresis. Comparison of the RNA species obtained from SalI-C with those produced from unfractionated DNA indicated that most of the RNAs and all of the major transcripts originate from the SalI C fragment; this fragment contains one copy of the terminally redundant portion of the genome. Seven major transcripts, a bidirectional terminator site, and 5 of the 13 minor transcripts were located within the 13-kilobase redundant region. The binding of polymerase to fragments of DNA produced by digestion of SalI-C with HpaII and HhaI was used to identify promoter-bearing regions within SalI-C.
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PMID:Analysis of bacteriophage SP82 major "early" in vitro transcripts. 626 Sep 90

We have examined the effect of the delta subunit on the interaction of the Bacillus subtilis RNA polymerase with an early gene promotor of phase SP82. Methylation by dimethyl sulfate, used to probe close approaches of polymerase to purines, revealed that noninitiated complexes formed by holo-enzyme (core-sigma-delta) had significantly fewer contacts than complexes formed by core-sigma. The presence or absence of delta had little or no effect on close approaches to purines in initiated complexes. DNAase I footprinting indicated that core-sigma was bound to the same region regardless of whether delta, initiating nucleotides, or both, were present. These data support the conclusion that delta acts prior to initiation to enhance promoter selectivity by limiting the number of possible interactions that the polymerase can make with DNA.
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PMID:The effect of the delta subunit on the interaction of Bacillus subtilis RNA polymerase with bases in a SP82 early gene promoter. 628 15


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