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)

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.
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PMID:Multiple proteins bind to VA RNA genes of adenovirus type 2. 356 5

RNA polymerase III faithfully transcribes the genes for ribosomal 5 S RNA, tRNA(1Met) or adenovirus VA RNA in vitro in the presence of required transcription factors. These genes display distinct differences in the kinetics of transcription complex formation and in their response to excess template. In contrast to tRNA and VA RNA synthesis, 5 S RNA synthesis displays a lag phase of 15 minutes before the onset of transcription and is clearly inhibited by high concentrations of template. Once formed, transcription complexes for the RNA polymerase III genes listed can be isolated by glycerol gradient centrifugation and display a remarkable stability against transient treatment with high salt concentrations. Complexes for 5 S RNA and tRNA remain functionally active up to 2.5 M-KCl. The activity of transcription complexes for VA RNA, however, is significantly diminished after treatment with high salt concentrations. This effect is shown to be due to an irreversible loss of transcription factors. RNA polymerase III is dissociated by high salt concentrations from all the transcription complexes studied but remains part of these complexes during the normal reinitiation cycle at 60 mM-KCl. An additional method for the purification of partial transcription complexes was developed that involves equilibrium centrifugation on cesium sulfate gradients. This method completely releases TFIIIB from 5 S complexes and a core complex, composed of the 5 S RNA gene, factors IIIA and IIIC, is retained. In the case of tRNA and VA RNA, core complexes are obtained that remain partly associated with TFIIIC and TFIIIB. These results indicate a qualitatively and/or quantitatively different interaction of individual factors in different polymerase III transcription complexes.
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PMID:Transcription complexes for various class III genes differ in parameters of formation and stability towards salt. 359 75

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.
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PMID:Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions. 378 24

We have performed in vitro kinetic analyses of transcription from the 5S gene promoter in order to resolve the rate-limiting events which lead to accurate transcription of the 5S RNA gene. We demonstrate that a rate-limiting intermediate can be formed during an extended incubation prior to initiation of transcription. Formation of such a complex is temperature-dependent, requires magnesium and ATP, consists of stoichiometric amounts of the known class III transcription factors and RNA polymerase III on 5S DNA, and eliminates the normal lag in attainment of a steady-state rate of transcription. This complex is therefore different from the "stable complex" minimally required for template commitment. Further analyses demonstrate that TFIIIB, like TFIIIA and TFIIIC, can be stably sequestered on the 5S gene and also allow us to formulate the following order of factor interactions on the 5S gene: TFIIIA, TFIIIC, TFIIIB, RNA polymerase III.
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PMID:Formation of a rate-limiting intermediate in 5S RNA gene transcription. 396 90

An analysis of mutant S. cerevisiae strains selected for their ability to increase transcription by RNA polymerase (pol) III has identified 14 isolates in which this phenotype is recessive. Genetic linkage and complementation studies suggest that all 14 isolates contain recessive alleles of PCF1. PCF1 encodes the 131-kDa subunit of transcription factor IIIC (TFIIIC131) and was identified previously by dominant mutations that also increased transcription by pol III. The recessive mutation, pcf1-3, results in a conservative substitution (R728-->K) towards the carboxyl-terminus of the protein. This position is distinct from the site of the dominant mutation PCF1-1 (H190-->Y), which maps to a tetratricopeptide repeat (TPR). Site-directed mutagenesis at amino acid 728 generated one allele, pcf1-4, with a stronger phenotype than pcf1-3. Extracts from pcf1-3 and pcf1-4 strains increase pol III transcription two- to threefold and ninefold, respectively, over wild-type under conditions that permit either single or multiple rounds of initiation. The entire effect of these mutations in vitro can be accounted for by an increase in the amount of transcriptionally active TFIIIB. In contrast, PCF1-1 primarily affects the rate of preinitiation complex assembly. The genetic, molecular, and biochemical data suggest that amino acid 728 in TFIIIC131 constitutes part of a structural domain in this protein that affects TFIIIB activity by influencing a previously undefined step in transcription. This step is suggested to occur after the recruitment of TFIIIB to DNA.
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PMID:Recessive mutations in the second largest subunit of TFIIIC suggest a new step in RNA polymerase III transcription. 748 59

The central RNA polymerase III (Pol III) transcription factor TFIIIB is composed of the TATA-binding protein (TBP), Brf, a protein related to TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembles autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a consensus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have examined how the binding polarity of TFIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is unable to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest preference for TFIIIB binding in the natural direction of the U6 promoter is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TFIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFIIIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specificity mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, the carboxy-terminal repeat of TBP contacts the upstream half of the TATA box. This orientation of yeast TBP in Pol III promoter-bound TFIIIB is the same as in Pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.
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PMID:The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB. 749 93

Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B". Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography. TBP and B" (apparent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detected between TBP and B", direct binding of [35S]-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA genes, there was no basal level of transcription in the complete absence of TBP. The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro. Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.
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PMID:Interactions between yeast TFIIIB components. 807 82

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable. A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.
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PMID:Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB. 756 18

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.
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PMID:Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains. 762 63

Two multisubunit complexes containing the TATA-binding protein (TBP) were isolated from HeLa cells constitutively expressing the FLAG epitope-tagged TBP using antibody affinity and peptide elution methods. One of the complexes (f:TFIID), isolated from the P11 0.85 M KCl fraction, contains at least 13 specific TBP-associated factors (TAFs) and can mediate activator-dependent transcription by RNA polymerase II. Importantly, activator function through the highly purified f:TFIID complex still requires a general cofactor fraction containing upstream factor stimulatory activity (USA). As previously observed with partially purified activator-competent natural TFIID, f:TFIID generates extended TATA-dependent footprints on the intrinsically strong adenovirus major late promoter (MLP) but only restricted footprints on the weak adenovirus E1b and E4 and HIV (core) promoters. Along with previous demonstrations of activator-induced downstream TFIID interactions on the E4 promoter, these results argue for a relationship between downstream interactions and overall promoter strength. Initiator-like sequences appear not to be essential for downstream interactions since they have no effect on downstream MLP interactions when mutated, do not effect downstream interactions on the HIV promoter and are not present on the inducible E4 promoter. The other multisubunit complex (f:TFIIIB), isolated from the P11 0.30 M KCl fraction, contains four specific TAFs and can substitute for one of the fractions (TFIIIB) required for RNA polymerase III (pol III) transcription. Neither f:TFIID nor TBP could substitute for this pol III TBP-containing fraction. This plus the fact that f:TFIIIB failed to generate a footprint on the MLP underscores the importance of TAFs in determining promoter specificity by different RNA polymerases.
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PMID:Unique TATA-binding protein-containing complexes and cofactors involved in transcription by RNA polymerases II and III. 768 40


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