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)

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

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.
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PMID:Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit. 198 83

Gene product 33 of phage T4 is known to be essential in late transcription. Upstream from gene 33 and overlapping its 5' terminal sequence by 20 bp, we identified an open reading frame coding for a binding protein for double-stranded DNA (DsbA). Gene product DsbA is composed of 89 amino acid residues with a Mr of 10376 kDa. We purified this protein to homogeneity from over-expressing cells. Gel retardation assays reveal that it binds to DNA and footprint analyses disclose that it interacts preferentially with T4 late promoter regions. At the sites of binding the protein introduces nicks in double-stranded DNA. In vitro transcription assays performed with T4 late modified RNA polymerase on restriction fragments harbouring a T4 late promoter region prove that gene product DsbA enhances transcription from these promoter regions in the presence of gene product 33. Gene dsbA is distinct from gene das which maps close to this genomic region.
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PMID:Gene product dsbA of bacteriophage T4 binds to late promoters and enhances late transcription. 201 38

The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.
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PMID:Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor. 211 6

Host cell RNA synthesis is inhibited by poliovirus infection. We have studied the mechanism of poliovirus-induced inhibition of RNA polymerase II-mediated transcription by using the adenovirus early region 3 (E3) promoter. In vitro transcription from the E3 promoter was severely inhibited in extracts prepared from poliovirus-infected HeLa cells. Four regions in the E3 promoter have been shown to serve as binding sites for cellular transcription factors. These regions contain binding sites for transcription factors NF-1 (site IV), AP-1 (site III), CREB/ATF (site II), and the TATA factor (site I). Binding to these four regions was not significantly altered by poliovirus infection as assayed by DNase I footprinting analysis; furthermore, gel retardation assays failed to reveal dramatic differences in the total amount of CREB/ATF-, AP-1-, and NF-1-binding activity present in mock- or poliovirus-infected cell extracts. Gel retardation assays, however, did reveal significant qualitative differences in the DNA-protein complexes formed with a CREB/ATF-binding site in extracts prepared from poliovirus-infected cells as compared to mock-infected cell extracts. Radioimmunoprecipitation reactions performed with antiserum against CREB/ATF revealed a severe reduction in a phosphorylated form of the protein present in poliovirus-infected cell extracts. However, in vitro kinase reactions demonstrated that mock- and poliovirus-infected cell extracts contained similar levels of CREB/ATF. Expression from the E3 promoter was shown to be activated by CREB/ATF in vivo; this induction was dependent upon the phosphorylation of CREB/ATF. Thus, we propose that poliovirus infection inhibits transcription from the E3 promoter, at least in part, through the dephosphorylation of CREB/ATF.
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PMID:Loss of a phosphorylated form of transcription factor CREB/ATF in poliovirus-infected cells. 216 27

We have identified a mitochondrial protein from Saccharomyces cerevisiae which confers the ability to recognize mitochondrial promoters onto a nonspecifically transcribing mitochondrial core RNA polymerase and we have purified this specificity factor 10,700-fold from a whole cell extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fraction followed by elution and renaturation of protein activity shows that the specificity factor is a 43-kDa polypeptide which directs mitochondrial core RNA polymerase to promoters belonging to rRNA-, tRNA-, and protein-encoding genes, as well as to mitochondrial replication origins. Gel filtration and glycerol gradient sedimentation studies indicate that the specificity factor shows little association with core RNA polymerase in the absence of DNA, and that it behaves like a monomeric 43-kDa protein.
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PMID:Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase. 244 67

The molecular basis for the greatly elevated expression of the cir gene (encoding the colicin I receptor) in cells unable to maintain a critical supply of intracellular iron was investigated by genetic and biochemical means. Deletion analysis of the cloned promoter region allowed delineation of sequences necessary for control of transcription initiating at the two promoters, P1 and P2. Gel retardation assays were used to demonstrate both binding of purified Fur (ferric uptake regulation) protein to the iron control region and lack of binding to DNA fragments which are not involved in cir regulation. An operator sequence spanning 43 to 47 base pairs and completely encompassing the two promoters was identified by DNase I protection experiments (footprinting), with binding occurring in a metal-dependent fashion. Thus, during iron-replete growth, Fur appears to act as a repressor of transcription by blocking formation of a DNA-RNA polymerase complex, analogous to the mechanism previously described for regulation of the aerobactin operon (V. de Lorenzo, S. Wee, M. Herrero, and J.B. Neilands, J. Bacteriol. 169:2624-2630, 1987). Characterized and putative Fur recognition sites from several genes were analyzed and classified by statistical methods.
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PMID:Mechanism for iron-regulated transcription of the Escherichia coli cir gene: metal-dependent binding of fur protein to the promoters. 264 21

An activity (designated BTF1Y) in extracts of Saccharomyces cerevisiae can substitute for the human TATA box-binding factor BTF1 in a reconstituted transcription system containing the adenovirus 2 major late promoter, RNA polymerase B (II), and the basic transcription factors BTF2, BTF3, and STF. We have purified BTF1Y to homogeneity, using as assays reconstitution of in vitro transcription and DNase I footprinting on the TATA element. Both activities copurified with a 27-kDa polypeptide as determined by SDS/PAGE. Gel filtration indicated a molecular mass of 28 +/- 5 kDa under nondenaturing conditions, suggesting that the native BTF1Y protein is a monomer. BTF1Y was enzymatically cleaved, several peptides were sequenced, and appropriate oligonucleotide probes were synthesized to clone the BTF1Y gene from a yeast genomic library. The BTF1Y gene contains a 720-base-pair open reading frame encoding a protein of 27,003 Da. The recombinant protein expressed in HeLa cells exhibited the same chromatographic characteristics and in vitro transcriptional activity as BTF1Y prepared from yeast extracts, confirming the identity of the gene. Gene-disruption experiments indicated that the yeast BTF1Y gene is a single-copy essential gene.
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PMID:Cloning of the gene encoding the yeast protein BTF1Y, which can substitute for the human TATA box-binding factor. 269 73

A set of internal deletions and frame-shift mutations was made in the structural gene for the major sigma factor of Escherichia coli RNA polymerase (sigma 70). The truncated proteins from these various mutants were examined to determine if they retained the ability to bind core RNA polymerase. Two assays were used to determine core-binding activity. Gel filtration was used to separate free sigma 70 from sigma 70 bound to core polymerase. Immunoprecipitation of polymerase using an anti-alpha-subunit monoclonal antibody was also used to determine if the various truncated proteins were bound to core. Results from these experiments indicate core-binding activity is retained when large portions of the sigma 70 protein are deleted. Deletion of a region in the central portion of the protein caused a large decrease in core-binding activity. The results suggest that the region spanning amino acids 361-390 is important for efficient core-binding activity. Sequence comparison of various sigma factors shows highly conserved amino acids in this region. A synthetic peptide having the sequence of amino acids 361-390 was synthesized and examined for the ability to bind core RNA polymerase.
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PMID:Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase. 269 3


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