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)

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

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.
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PMID:Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators. 759 78

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAFII complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system. We have cloned and sequenced the genes encoding four of the multiple yeast TAFII proteins comprising the TBP-TAFII multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFII complex is minimally composed of TBP and seven distinct yTAFII proteins ranging in size from M(r) = 150,000 to M(r) = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability.
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PMID:Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae. 766 72

TATA-binding protein (TBP)-associated factors (TAFs) in TFIID are required for activator proteins to stimulate transcription, but the mechanism by which TAFs function is poorly understood. To study how TAFs participate in transcriptional activation by the Epstein-Barr virus activator Zta, we used agarose gel electrophoresis and DNase I footprinting to compare transcription complex assembly in reactions with either TFIID or TBP in the presence and absence of wild-type Zta or a deletion of Zta lacking its activation domain. A stable complex of promoter DNA with Zta, TFIIA, and TFIID rapidly formed on a template with Zta-binding sites. Zta stimulation of stable complex formation required TAFs as well as the Zta activation domain and TFIIA. The Zta activation domain also induced a TAF-dependent DNA-protein interaction near and downstream of the transcription star site. Stable complexes formed within 1 min supported activated transcription when RNA polymerase II and the remaining general transcription factors were subsequently added. This rapid assembly of a stable Zta-TFIIA-TFIID-promoter complex is probably a significant component of the mechanism by which TAFs and the Zta activation domain cooperate to stimulate transcription.
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PMID:A mechanism for TAFs in transcriptional activation: activation domain enhancement of TFIID-TFIIA--promoter DNA complex formation. 792 93

The multisubunit transcription factor TFIID is an essential component of the RNA polymerase II initiation apparatus. Recent studies suggest that TFIID subunits, or TAFs associated with the TATA-binding protein (TBP), play a critical role in modulating transcriptional activation by sequence-specific DNA-binding factors. Thus far, six of the largest TAFs associated with Drosophila TFIID have been cloned and partially characterized. Here, we report the molecular cloning, expression, and subunit interaction specificities of two small molecular mass TAFs. Both dTAFII30 alpha and dTAFII30 beta are associated with TFIID via interactions with other TAFs, including dTAFII250, dTAFII150, and dTAFII110. In addition, dTAFII30 alpha also contacts dTBP. The carboxy-terminal half of dTAFII110 was found to contact a short 67-amino-acid region of dTAFII30 alpha, which is predicted to form two potential alpha-helices, one of which is amphipathic. Interestingly, dTAFII30 alpha also appears to multimerize through its carboxy-terminal region. Although neither dTAFII30 alpha nor dTAFII30 beta have been found to interact with specific activators thus far, it is intriguing that both bind other TAFs such as dTAFII110 and dTAFII150, which are the targets of activation domains. Our studies suggest that both of the small subunits of TFIID play a role in the assembly of the complex and may contribute to the stability of multiple TAF-TAF interactions.
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PMID:Molecular cloning and characterization of dTAFII30 alpha and dTAFII30 beta: two small subunits of Drosophila TFIID. 827 41

The TATA-binding proteins (TBP) from both human and Drosophila have been shown to exist in various distinct multiprotein complexes that are required, respectively, for transcription by all three RNA polymerases. In contrast, in vitro biochemical analyses have suggested that yeast TBP exists as a monomeric 27-kDa protein free in solution. We have examined the oligomerization state of yeast TBP and report here that yeast TBP, like human and Drosophila TBPs, is also stably associated with other proteins in vitro. Using anti-TBP antibodies we have immunopurified yeast TBP and associated factors (TBP-associated factors or TAFs). When this fraction was analyzed by SDS-polyacrylamide gel electrophoresis, polypeptides of approximate relative molecular size ranging from 170 to 60 kDa are prominently represented. Immunoblot analysis revealed that one of these TAFs, TAF70, corresponds to BRF1/TDS4/PCF4, a subunit of transcription factor (TF) IIIB. Furthermore, this highly purified TAF fraction can reconstitute polymerase III transcription when supplemented with purified RNA polymerase III and TFIIIC. Our data indicate that our TAF fraction contains TFIIIB transcription factor activity and that all the subunits of yeast TFIIIB are stably complexed with TBP.
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PMID:Immunopurification of yeast TATA-binding protein and associated factors. Presence of transcription factor IIIB transcriptional activity. 834 Mar 60

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.
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PMID:In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive. 860 57

An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF-IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species-specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB-directed initiation complexes at the murine rDNA promoter.
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PMID:Species specificity of ribosomal gene transcription: a factor associated with human RNA polymerase I prevents transcription of mouse rDNA. 863 44

We describe the cloning and analysis of TAF25, a previously uncharacterized yeast gene that encodes a yeast TATA-binding protein-associated factor or yTAF of Mr = 25,000. The gene encoding yTAF25 is a single copy essential gene, and the protein sequence deduced from TAF25 exhibits sequence similarity to a metazoan hTAFII. The results from immunological studies confirm that yTAF25 is a subunit of a large multiprotein TATA-binding protein-yeast TATA-binding protein-associated factor complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Both genetic and biochemical analyses demonstrate that yTAF25 can interact directly with itself. Transcriptional data show that the activity of the multiprotein complex containing yTAF25 is RNA polymerase II-specific, thus indicating that TAF25 encodes a bona fide yeast RNA polymerase II TAF. Hence the protein encoded by TAF25 has been termed yTAFII25.
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PMID:Isolation and characterization of TAF25, an essential yeast gene that encodes an RNA polymerase II-specific TATA-binding protein-associated factor. 866 25

An unusual property of ribosomal gene transcription is its marked species specificity. This results from distinct promoter-recognition properties of the RNA polymerase I transcription apparatus. The purification and functional characterization of TIF-IB/SL1, a promoter-recognition factor containing the TATA-binding protein, as well as the recent cloning of cDNAs encoding the three subunits (TAF(I)s) of the respective human and mouse factor, will facilitate the molecular analysis of the mechanisms underlying species-specific rDNA transcription and reveal how the basal transcriptional machinery has evolved.
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PMID:Species specificity of transcription by RNA polymerase I. 866 54


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