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)

We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of PR result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which PR sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the PR promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the PR promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the PR promoter.
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PMID:RNA polymerase bound to the PR promoter of bacteriophage lambda inhibits open complex formation at the divergently transcribed PRM promoter. Implications for an indirect mechanism of transcriptional activation by lambda repressor. 183 35

Expression of the p53 gene plays an important role in the regulation of cellular proliferation and malignant transformation. Overexpression of mutant forms of p53 is in fact a common feature of many transformed cells. Studies dealing with the transcriptional regulatory regions of the p53 gene indicate that, unlike most promoters transcribed by RNA polymerase II, the p53 promoter contains no TATA-like sequence upstream of the transcription start site. Here we demonstrate that the murine p53 promoter contains a cis-acting element that maps downstream to the transcription initiation site. The integrity of this element is required for high-level expression from the promoter in transformed cells. By DNase I protection and mobility-shift analysis, we show that a nuclear factor binds to this downstream element through the consensus recognition sequence for the helix-loop-helix (HLH)-containing proteins of the myc/MyoD family of transcriptional regulators. We propose that the activity of one or more members of this family of transcription factors is an important determinant in the expression of p53 and that at least one level of p53 overexpression in transformed cells may thus be due to aberrant expression of the relevant factor(s). Furthermore, the possibility that the regulation of expression of p53 occurs, in part, by means of a potential HLH-containing factor provides a possible mechanism for the suppression of proliferation by the MyoD family of transcriptional regulators.
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PMID:Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif. 185 94

We have demonstrated earlier that human cells contain nuclear protein interacting with conserved GC-rich sequence motifs of human Alu-family DNA repeats. One of these sequences is located in the region between elements A and B of bipartite RNA polymerase III promoter of Alu (AB-region). In this study we have used a DNase I footprinting assay with an Alu restriction subfragment covering AB-region, as well as a gel mobility shift assay with appropriate synthetic oligonucleotides to analyse in more detail the interaction of the protein with AB-region. We have also used antibodies raised against a zinc-finger peptide to examine the presence of a zinc-finger in the Alu-binding protein. The results indicate that AGG triplets may be important for high-affinity binding of the protein to DNA, and that the Alu-binding protein is a zinc-finger protein.
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PMID:Binding specificity of human nuclear protein interacting with the Alu-family DNA repeats. 185 21

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.
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PMID:Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli. 186 Aug 20

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
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PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57

Vaccinia virus RNA polymerase requires the heterodimeric protein, vaccinia early transcription factor (VETF), for transcription of early gene templates in vitro. We have analyzed the vaccinia growth factor promoter sequences interacting with VETF at the nucleotide level and provide evidence that the factor contacts the DNA at two separate sites. DNase I protection analysis showed that VETF was found to nucleotides -12 to -29 relative to the transcription initiation site, and also to nucleotides +8 to +10 downstream of the initiation site. The importance of both binding sites for stable complex formation was supported by methylation interference analysis. Using synthetic oligonucleotides encoding different parts of the vaccinia growth factor promoter, it was shown that nucleotides down-stream of the transcription initiation site are required for stable complex formation. Competition binding experiments demonstrated that only the upstream binding site contributes significantly to binding specificity. Binding to two separated DNA sequences results in a bend in the promoter DNA as demonstrated by electrophoretic mobility shift analysis of permuted DNA fragments. These findings suggest that VETF activates transcription by sequence specific binding and structural alteration of the promoter DNA helix.
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PMID:Promoter DNA contacts made by the vaccinia virus early transcription factor. 186 71

RNA polymerase III transcription factor TFIIIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of 90 and 70 kDa, that were previously identified by photocrosslinking to DNA. It is shown here that TFIIIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription. DNA-protein photocrosslinking experiments show these two components separately contain the 90- and 70-kDa TFIIIB-specific polypeptides. The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIIIC and DNA, stabilizes TFIIIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIIIB. The 90-kDa component of TFIIIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIIB and bestows heparin-insensitivity on the TFIIIB-DNA complex. The resolution of TFIIIB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.
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PMID:Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties. 187 Nov 37

Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma A-associated RNA polymerase (EC 2.7.7.6). The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form o RNA polymerase containing sigma A, the primary sigma factor in vegetative cells. The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We found that single-base-pair substitutions in the around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity. DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro. Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma A-recognition sequence. These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation.
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PMID:Spo0A binds to a promoter used by sigma A RNA polymerase during sporulation in Bacillus subtilis. 190 44

We have developed an in vitro transcription system in which purified TrpI protein and indoleglycerol phosphate (InGP) activate transcription initiation at the trpBA promoter (trpPB) and repress initiation at the trpI promoter (trpPI) of Pseudomonas aeruginosa. The phenotypes resulting from mutations in the -10 region of both promoters indicate that the -10 region consensus sequence in P. aeruginosa is probably the same as that in Escherichia coli. Furthermore, in the absence of TrpI and InGP, the activities of the two promoters are inversely correlated: down mutations in trpPI lead to increased activity of trpPB, and up mutations in trpPB cause a decrease in trpPI activity. These results are a consequence of the fact that the two promoters overlap, so that RNA polymerase cannot form open complexes with both promoters simultaneously. Thus, in theory, by preventing RNA polymerase from binding at trpPI, TrpI protein could indirectly activate trpPB. However, oligonucleotide-induced mutations that completely inactivate trpPI do not relieve the requirement for TrpI and InGP to activate trpPB. Therefore, activation of trpPB is mediated by a direct effect of TrpI on transcription initiation at trpPB. In addition, the oligonucleotide-induced mutations in trpPI alter site II, the weaker of two TrpI binding sites identified in DNase I and hydroxyl radical footprinting studies (M. Chang and I. P. Crawford, Nucleic Acids Res. 18:979-988, 1990). Since these mutations prevent full activation of trpPB, we conclude that specific base pairs in site II are required for activation.
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PMID:Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro. 190 58

Protein p4 of the Bacillus subtilis phage phi 29 switches on the transcription of the viral late genes by binding to the viral late promoter at a region close to the RNA polymerase binding site. Gel retardation and DNase I footprinting assays show that the presence of protein p4 is required for RNA polymerase recognition of the late promoter. The protein p4 and RNA polymerase DNA binding sites have been separated by the insertion of bent and non-bent DNA sequences of different lengths. These mutant promoters were used to study in vitro their protein p4-dependent transcriptional activity and their interaction with both protein p4 and RNA polymerase. The results indicate that protein p4 is able to function at longer DNA distances from the RNA polymerase binding site than in the natural promoter. The extent of protein p4 activity depended on the length and conformation of the inserted DNA. Activation of transcription and RNA polymerase binding was favoured when the relative orientation of protein p4 and RNA polymerase was conserved and when the intervening DNA had a bent conformation. These data, together with the DNase I footprints, suggest that activation at distance by protein p4 involves a DNA loop held by the interaction of protein p4 and RNA polymerase.
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PMID:Transcription activation at a distance by phage phi 29 protein p4. Effect of bent and non-bent intervening DNA sequences. 190 41


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