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)

An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.
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PMID:Two distinct transcriptional activities of pea (Pisum sativum) chloroplasts share immunochemically related functional polypeptides. 141 45

A dominant mutation in the PCF4 gene of S. cerevisiae was isolated as a suppressor of a tRNA gene A block promoter mutation. In vitro studies indicate that PCF4 is a stoichiometrically-required RNA polymerase III (pol III) transcription initiation factor. We show that the PCF4-1 mutation increases the number of transcriptionally competent preinitiation complexes by affecting a limiting activity in yeast cell extracts that is squelched by excess TFIIIC. The PCF4 gene encodes a TFIIB homolog whose size, biochemical, and genetic properties are consistent with those of the 70 kd subunit of TFIIIB. The TFIIB homology of PCF4 suggests a means for determining the polymerase specificity of a gene.
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PMID:PCF4 encodes an RNA polymerase III transcription factor with homology to TFIIB. 142 89

Double-stranded RNA viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A double-stranded RNA virus we have investigated the mechanism by which the virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag-pol fusion protein which is formed by a -1 ribosomal frameshift. Here we show that Gag alone is sufficient for virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A virus encapsidation model: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.
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PMID:Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus. 143 38

The TATA-binding protein (TBP) is required for transcription by RNA polymerase III (pol III), even though many pol III templates, such as the adenovirus VA1 gene, lack a consensus TATA box. We show that TBP alone does not form a stable, productive interaction with VA1 DNA. However, it can be incorporated into an initiation complex if the other class III basal factors, TFIIIB and TFIIIC, are also present. TFIIIB can associate with the evolutionarily conserved C-terminal domain of TBP in the absence of DNA or TFIIIC, suggesting that TFIIIB exists in solution as a complex with TBP. The stable association of TBP with an essential component of the pol III transcription apparatus may account for the ability of TATA-less class III genes to recruit TBP.
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PMID:Mechanism of TATA-binding protein recruitment to a TATA-less class III promoter. 145 35

We have investigated the requirement for TBP (TATA-binding protein) in transcription mediated by RNA polymerase III (pol III) in fractionated HeLa cell extracts. Two activities, TFIIIB and TFIIIC, found in phosphocellulose fractions PC B and PC C respectively, have been defined as necessary and sufficient, with pol III, for in vitro transcription of tRNA genes. Depletion of TBP from PC B, using antibodies raised against human TBP, is shown to inhibit the pol III transcriptional activity of the fraction. Furthermore, TBP is present in fractions with human TFIIIB activity, and a proportion of TBP cofractionates with TFIIIB over four chromatographic purification steps. TFIIIB fractions are capable of supplying TBP in the form necessary for pol III transcription, and cannot be substituted by fractions containing other TBP complexes or TBP alone. The use of a 5S RNA gene and two tRNA templates supports the general relevance of our findings for pol III gene transcription. Purified TFIIIB activity can also support pol II-mediated transcription, and is found in a complex of approximately 230kD, suggesting that TFIIIB may be the same as the previously characterized B-TFIID complex (1,2). We suggest that transcription by the three RNA polymerases is mediated by distinct TBP-TAF complexes: SL1 and D-TFIID for pol I and pol II respectively, and TFIIIB for pol III.
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PMID:Cofractionation of the TATA-binding protein with the RNA polymerase III transcription factor TFIIIB. 146 21

In a reconstituted system consisting of partially purified RNA polymerase I (pol I) and the initiation factors TIF-IA, TIF-IB, and TIF-IC, the nucleolar factor UBF (upstream binding factor) stimulates transcription from the rRNA-encoding DNA (rDNA) promoter at least 50-fold. This activation is not observed at high template concentrations or in the presence of highly purified pol I. Template commitment experiments suggest that UBF activates transcription by relieving inhibition exerted by a negative-acting factor(s) in the polymerase fraction that competes for TIF-IB binding to the rDNA promoter and prevents the formation of preinitiation complexes. Using purified histone H1 bound to DNA as a model for the repressed state of the rDNA promoter, we show that UBF counteracts H1-mediated repression of pol I transcription. The implications of these findings are discussed with respect to the protein-protein and protein-DNA interactions at the rDNA promoter and the possible involvement of UBF in control of ribosomal gene transcription.
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PMID:Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor. 150 43

Premature termination of transcription is assumed to be an important mechanism of c-myc regulation. Induction of terminal differentiation in the promyelocytic leukemia cell line HL60 by dimethyl-sulfoxide (DMSO) is accompanied by a block of RNA elongation within the first exon of the c-myc gene. We have studied the 3'-structure of incompletely elongated transcripts in (i) nuclear RNA of induced and uninduced HL60 cells, (ii) nuclear run-on RNA, and (iii) RNA of in vitro transcribed c-myc constructs. Elongation of c-myc RNA stopped in all three transcriptional systems at similar sites distributed 150-350 bases downstream of the P2 promoter. When HL60 cells were induced to terminal differentiation the short c-myc exon 1 specific RNAs disappeared in nuclear RNA. This implied that RNA polymerase II (pol II) does not continue to transcribe c-myc exon 1 in induced HL60 cells as suggested by earlier nuclear run-on experiments. Therefore, kinetic experiments with small oligonucleotides as probes were performed to determine the start position of pol II on c-myc exon 1 in nuclear run-ons. The results demonstrate that all RNA polymerases are localized at the c-myc P2 promoter in DMSO-treated HL60 cells. Preparation of nuclei for run-on experiments induces a release of pol II from the c-myc P2 promoter leading to the strong nuclear run-on signal on c-myc exon 1. Thus, down-regulation of c-myc in differentiating HL60 cells occurs by retention of pol II at the transcription start site.
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PMID:Hold back of RNA polymerase II at the transcription start site mediates down-regulation of c-myc in vivo. 150 20

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3'-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to alpha-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.
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PMID:Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III. 151 Nov 42

The assembly of activated RNA polymerase II (pol II) transcription complexes has been investigated by assaying whether pre-assembly of intermediate complexes reduces the extended time required for start-site melting. The results show that a closed complex requiring factors IIA, IID, and the acidic activator GAL4-AH forms in a rate-limiting step. This directs the templates into a productive assembly pathway. Factor TFIIB is then added rapidly, affording further protection against diversion into nonproductive pathways. These events are followed by a series of rapid steps in which the remaining general factors are assembled onto the template, which is then melted using the energy of ATP hydrolysis.
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PMID:The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA. 151 30

The L-A double-stranded RNA virus of Saccharomyces cerevisiae makes a gag-pol fusion protein by a -1 ribosomal frameshift. The pol amino acid sequence includes consensus patterns typical of the RNA-dependent RNA polymerases (EC 2.7.7.48) of (+) strand and double-stranded RNA viruses of animals and plants. We have carried out "alanine-scanning mutagenesis" of the region of L-A including the two most conserved polymerase motifs, SG...T...NT..N (. = any amino acid) and GDD. By constructing and analyzing 46 different mutations in and around the RNA polymerase consensus regions, we have precisely defined the extent of domains and specific residues essential for viral replication. Assuming that this highly conserved region has a common secondary structure among different viruses, we predict a largely beta-sheet structure.
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PMID:RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains. 154 80


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