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)

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

The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.
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PMID:Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein. 750 37

Fractions obtained from HeLa cell extracts were used to study RNA polymerase III-catalyzed transcription from the human 7SK and mouse U6 RNA promoters in vitro. Although both genes depend on two almost identical core promoter elements (TATA box and PSE), different fractions were required. The 7SK promoter revealed full activity with the phosphocellulose B fraction alone. In contrast, efficient transcription from the U6 promoter depended on the additional presence of the C or D fraction. The analysis of the b1 and b2 subfractions (obtained by DEAE-Sephadex chromatography) revealed that for both promoters the b1 and the phosphocellulose D fraction were mutually interchangeable. However, while both fractions were fully equivalent for the 7SK promoter, the U6 promoter revealed an additional requirement for the C fraction in the presence of the b1 fraction. Since the b1 and the D fractions enclose two different complexes of the TATA-binding protein (TBP), B-TFIID and D-TFIID, our results indicate that functionally these two complexes are responsible for the observed differences in transcription of the 7SK and U6 genes.
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PMID:The seemingly identical 7SK and U6 core promoters depend on different transcription factor complexes. 750 70

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

The human T-lymphotropic virus type I (HTLV-I) promoter contains the structural features of a typical RNA polymerase II (pol II) template. The promoter contains a TATA box 30 bp upstream of the transcription initiation site and binding sites for several pol II transcription factors, and long poly(A)+ RNA is synthesized from the integrated HTLV-I proviral DNA in vivo. Consistent with these characteristics, HTLV-I transcription activity was reconstituted in vitro by using TATA-binding protein, TFIIA, recombinant TFIIB, TFIIE, and TFIIF, TFIIH, and pol II. Transcription of the HTLV-I promoter in the reconstituted system requires RNA pol II. In HeLa whole cell extracts, however, the HTLV-I long terminal repeat also contains an overlapping transcription unit (OTU). HTLV-I OTU transcription is initiated at the same nucleotide site as the RNA isolated from the HTLV-I-infected cell line MT-2 but was not inhibited by the presence of alpha-amanitin at concentrations which inhibited the adenovirus major late pol II promoter (6 micrograms/ml). HTLV-I transcription was inhibited when higher concentrations of alpha-amanitin (60 micrograms/ml) were used, in the range of a typical pol III promoter (VA-I). Neutralization and depletion experiments with three distinct pol II antibodies demonstrate that RNA pol II is not required for HTLV-I OTU transcription. Antibodies to basal transcription factors TATA-binding protein and TFIIB, but not TFIIIC, inhibited HTLV-I OTU transcription. These observations suggest that the HTLV-I long terminal repeat contains overlapping promoters, a typical pol II promoter and a unique pol III promoter which requires a distinct set of transcription factors.
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PMID:Transcription of the human T-cell lymphotropic virus type I promoter by an alpha-amanitin-resistant polymerase. 752 15

Transcription factor IIB (TFIIB) plays a central role in the assembly of the RNA polymerase II initiation complex. Monoclonal antibodies (mAbs) that react with human TFIIB were prepared and used as probes to identify portions of TFIIB that are accessible when the factor is in solution and when it is contained in a complex with DNA. Seven mAbs were examined and were mapped to three regions of the TFIIB molecule. Only the mAbs that mapped to residues 52-105 inhibited transcription, immunoprecipitated recombinant TFIIB and TFIIB from HeLa cell nuclear extract (NE), and supershifted a complex containing TFIIB, the TATA-binding protein, and DNA. The mAbs that mapped to residues 1-51 and the mAb that mapped to residues 106-316 did not show activity in the functional assays, with the exception of the far N-terminal mAbs (residues 1-51), which immunoprecipitated recombinant TFIIB, but not TFIIB from HeLa cell NE. These data indicate that the region containing residues 52-105 is exposed in solution and when TFIIB is part of the preinitiation complex and that some far N-terminal epitopes are accessible on the purified protein, but become blocked when TFIIB is in HeLa cell NE or in the preinitiation complex.
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PMID:Accessibility of epitopes on human transcription factor IIB in the native protein and in a complex with DNA. 753 65

The TATA-binding protein (TBP) contains a concave surface that interacts specifically with TATA promoter elements and a convex surface that mediates protein-protein interactions with general and gene-specific transcription factors. Biochemical experiments suggest that interactions between activator proteins and TBP are important in stimulating transcription by the RNA polymerase II machinery. To gain insight into the role of TBP in mediating transcriptional activation in vivo, we implemented a genetic strategy in Saccharomyces cerevisiae that involved the use of a TBP derivative with altered specificity for TATA elements. By genetically screening a set of TBP mutant libraries that were biased to the convex surface that mediates protein-protein interactions, we identified TBP derivatives that are impaired in the response to three acidic activators (Gcn4, Gal4, and Ace1) but appear normal for constitutive polymerase II transcription. A genetic complementation assay indicates that the activation-defective phenotypes reflect specific functional properties of the TBP derivatives rather than an indirect effect on transcription. Surprisingly, three of the four activation-defective mutants affect residues that directly contact DNA. Moreover, all four mutants are defective for TATA element binding, but they interact normally with an acidic activation domain and TFIIB. In addition, we show that a subset of TBP derivatives with mutations on the DNA-binding surface of TBP are also compromised in their responses to acidic activators in vivo. These observations suggest that interactions at the TBP-TATA element interface can specifically affect the response to acidic activator proteins in vivo.
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PMID:Mutations on the DNA-binding surface of TATA-binding protein can specifically impair the response to acidic activators in vivo. 756 97

The binding of TATA-binding protein (TBP) to the TATA element is the first step in the initiation of RNA polymerase II transcription from many promoters in vitro. It has been proposed that upstream activator proteins stimulate transcription by recruiting TBP to the promoter, thus facilitating the assembly of a transcription complex. However, the role of activator proteins acting at this step to stimulate transcription in vivo remains largely speculative. To test whether recruitment of TBP to the promoter is sufficient for transcriptional activation in vivo, we constructed a hybrid protein containing TBP of the yeast Saccharomyces cerevisiae fused to the DNA-binding domain of GAL4. Our results show that TBP recruited by the GAL4 DNA-binding domain to promoters bearing a GAL4-binding site can interact with the TATA element and direct high levels of transcription. This finding indicates that binding of TBP to promoters in S. cerevisiae is a major rate-limiting step accelerated by upstream activator proteins.
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PMID:Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator. 756 28

The promoter of vertebrate U6 small nuclear RNA genes consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III transcription. We detected RNA polymerase II transcription as well as pol III transcription from the human U6 promoter in a HeLa nuclear extract. The pol II-specific transcription was independent of the PSE and dependent on the presence of the TATA box. Both pol III- and pol II-specific transcription were stimulated by addition of recombinant TATA-binding protein (TBP). We conclude that both pol III and pol II preinitiation complexes can assemble on the U6 promoter in vitro and could compete during the bona fide process in the cell.
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PMID:Both RNA polymerase III and RNA polymerase II accurately initiate transcription from a human U6 promoter in vitro. 757 66

The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria). Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya. For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes. Here, we report the cloning of a Sulfolobus shibatae gene that encodes a protein (transcription factor TFB) with striking homology to the eukaryotic basal transcription factor TFIIB. We show by primer extension analysis that transcription of the S. shibatae TFB gene initiates 27 bp downstream from a consensus A-box element. Significantly, S. shibatae TFB contains an N-terminal putative metal-binding region and two imperfect direct repeats--structural features that are well conserved in eukaryotic TFIIBs. This suggests that TFB may perform analogous functions in Archaea and Eucarya. Consistent with this, we demonstrate that S. shibatae TFB promotes the binding of S. shibatae TBP to the A-box element of the Sulfolobus 16S/23S rRNA gene. Finally, we show that S. shibatae TFB is significantly more related to TFB of the archaeon Pyrococcus woesei than it is to eukaryotic TFIIBs. These data suggest that TFB arose in the common archaeal/eukaryotic ancestor and that the lineages leading to P. woesei and S. shibatae separated after the divergence of the archaeal and eukaryotic lines of descent.
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PMID:Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae. 759 84


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