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Query: UNIPROT:P20226 (TATA-binding protein)
 1,297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A T7 RNA polymerase expression system has been used for the efficient expression of the yeast RNA polymerase general transcription factor TFIID (TFIIDY), the TATA-box factor (previously called BTF1) in Escherichia coli. Expression of the gene was performed at 25 degrees C instead of 37 degrees C to increase the total amount of soluble TFIIDY. Soluble TFIIDY was purified in three chromatographic steps and was eluted from the final column, a heparin-5PW HPLC column, in two peaks at 0.38 M (peak I) and 0.42 M (peak II) KCl in which this protein was 52% and greater than 95% pure, respectively. The protein in both peaks was active in an in vitro transcription assay. However, while TFIIDY from peak II was essentially indistinguishable from the material isolated from yeast, the protein of peak I differed in a number of biochemical characteristics, having a lower specific activity in an in vitro transcription assay and displaying an altered pattern of bands in a DNA band shift assay. Despite these differences, the proteins in both peaks have identical molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable N-terminal amino acid sequences, and apparently exist as monomers under the conditions used for the heparin-5PW chromatography.
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PMID:Expression in Escherichia coli: purification and properties of the yeast general transcription factor TFIID. 182 18

The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.
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PMID:Yeast TATA-box transcription factor gene. 268 26

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.
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PMID:Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes. 789 97

The transforming proteins encoded by the adenovirus E1A gene bind to a 300-kDa cellular product, p300, via the N-terminal E1A sequences. Residues important for p300 binding are required for the transformation function of E1A and for other E1A-mediated gene-regulating functions, including activation of cell cycle-regulated products and repression of tissue-specific enhancer activity. Recent evidence indicates that p300 is a DNA-binding protein with specific affinity for known enhancer motifs, suggesting that p300 may be a component of transcription factor complexes. The possibility that upstream element-binding factors might interact with basal transcription factors led us to investigate whether p300 interacts, directly or indirectly, with the TATA-binding protein (TBP). We report here that TBP-specific immunoprecipitations show a 300-kDa protein co-precipitating with TBP. This protein is lost from the precipitated material if the lysates are boiled in sodium dodecyl sulfate prior to immunoprecipitation, implying that its presence does not result from non-specific antibody cross-reactivity, but is dependent on specific association with TBP. The TBP-associated 300-kDa protein and p300 originally defined by E1A association show indistinguishable partial proteolytic digest patterns, indicating that these are identical or closely related species. Moreover, p300-specific complexes and TBP-specific complexes include at least two additional common polypeptide species, phosphoproteins of 64 and 59 kDa. These results suggest that p300 interacts with TBP, possibly through intermediate protein-protein associations. They thus provide additional biochemical evidence for postulated protein-protein interactions between upstream regulatory factors and the basal transcriptional machinery.
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PMID:p300, and p300-associated proteins, are components of TATA-binding protein (TBP) complexes. 850 84

Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UBF from rat cell nuclear extracts and partially purified SL-1 from human cells coimmunoprecipitated with the anti-UBF antibody after mixing, indicating that the UBF-SL-1 complex can re-form. Treatment of UBF-depleted extracts with the anti-UBF antibody depleted the extracts of SL-1 activity only if UBF was added to the extract prior to the immunodepletion reaction. Furthermore, SL-1 activity could be recovered in the immunoprecipitate. Interestingly, these immunoprecipitates did not contain RNA polymerase I, as a monospecific antibody to the 194-kDa subunit of RNA polymerase I failed to detect that subunit in the immunoprecipitates. Treatment of N1S1 cell extracts with the anti-UBF antibody depleted the extracts of SL-1 activity but not TFIIIB activity, suggesting that the binding of UBF to SL-1 is specific and not solely mediated by an interaction between UBF and TATA-binding protein, which is also a component of TFIIIB. These data provide evidence that UBF and SL-1 interact.
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PMID:The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor. 855 83

Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of approximately 2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.
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PMID:Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription. 985 2

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.
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PMID:A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding. 1178 52

The TATA-binding protein (TBP) plays a central role in the assembly of most eukaryotic transcription initiation complexes. We have characterized 3 monoclonal antibodies (mAbs) that react in the far amino-terminal (N-terminal) domain of the human TBP molecule (residues 1-99). One of these mAbs (designated 1TBP22) is a polyol-responsive monoclonal antibody (PR-mAb) and was adapted to an immunoaffinity chromatography procedure for purifying bacterially expressed, recombinant human TBP. The epitope for mAb 1TBP22 maps to residues 55-99, which includes the polyglutamine region. However, mAb 1TBP22 does not react with poly-l-glutamine. Human TBP, contained on the pET11a plasmid, was expressed in Escherichia coli Rosetta (DE3)pLysS. The cell lysate from 330 ml of induced culture was treated with polyethyleneimine (PEI) at 0.5 M NaCl to precipitate the nucleic acids. After centrifugation, the supernatant fluid was applied to an immunoadsorbent containing mAb 1TBP22. After extensive washing, the TBP was eluted with buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. Human TPB purified by the immunoaffinity chromatography method was found to be active in gel-shift assays and transcription assays. Preliminary data indicate that this mAb might be useful for purifying protein complexes containing TBP from HeLa cell extracts.
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PMID:Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaffinity chromatography. 1524 40