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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 Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".
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PMID:The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. 145 36

The gene encoding yeast U6 snRNA that is transcribed by RNA polymerase III (Pol III) contains both a TATA box upstream of the transcription start site and a downstream binding site for the factor TFIIIC. This juxtaposition of elements typical of both Pol II- and Pol III-transcribed genes raises the question of how polymerase specificity is determined. The upstream U6 promoter containing the TATA box and transcription start site was shown previously to be transcribed by Pol III in vitro. We therefore tested whether the upstream promoter of yeast U6 encodes Pol III specificity. One model is that polymerase specificity is conferred by the homologous Pol II and Pol III transcription factors TFIIB and BRF1. However, we found no specificity in the binding of BRF1 or TFIIB to TATA-containing promoters of genes specifically transcribed by Pol III or Pol II. Yeast strains deficient for Pol II or Pol III transcription were employed to examine U6 polymerase specificity in vivo. We find that the U6 upstream promoter is Pol II-specific in vivo and is converted to Pol III specificity by TFIIIC. Thus, preferential recruitment of TFIIIB by TFIIIC probably excludes the Pol II general factors and promotes Pol III transcription, thereby determining polymerase specificity.
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PMID:TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter. 770 60

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

The yeast RNA polymerase III (pol III) general transcription factor TFIIIB is composed of three subunits; the TATA-binding protein (TBP)1, the TFIIB-related factor (BRF1), and a third factor termed TFIIIB90 or B". Here we report the purification of yeast TFIIIB90, cloning of the gene encoding TFIIIB90, and reconstitution of TFIIIB from recombinant polypeptides. The TFIIIB90 open reading frame encodes a 68-kDa polypeptide and has no obvious similarity to any other known protein sequences. The gene encoding TFIIIB90 is essential for viability of yeast. Using recombinant TFIIIB subunits, we found that TFIIIB90 interacts weakly with TBP in the absence of BRF1, and that this interaction is enhanced at least 25-fold by BRF1. In addition, TFIIIB90 showed pol III specificity as it could not interact with the pol II-specific TFIIB-TBP-DNA complex. To localize the regions of the TBP-DNA complex that interact with BRF1 and TFIIIB90, we tested whether the pol II factors TFIIA and TFIIB interfered with the binding of BRF1 and TFIIIB90 to TBP-DNA. Our results suggest that the binding sites for BRF1 and TFIIIB90 on TBP-DNA both overlap the binding sites for TFIIA and TFIIB.
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PMID:Cloning and functional characterization of the gene encoding the TFIIIB90 subunit of RNA polymerase III transcription factor TFIIIB. 866 56

Transcription factor IIIB (TFIIIB) is directly involved in transcription initiation by RNA polymerase III in eukaryotes. Yeast contain a single TFIIIB activity that is comprised of the TATA-binding protein (TBP), TFIIB-related factor 1 (BRF1), and TFIIIB", whereas two distinct TFIIIB activities, TFIIIB-alpha and TFIIIB-beta, have been described in human cells. Human TFIIIB-beta is required for transcription of genes with internal promoter elements, and contains TBP, a TFIIIB" homologue (TFIIIB150), and a BRF1 homologue (TFIIIB90), whereas TFIIIB-alpha is required for transcription of genes with promoter elements upstream of the initiation site. Here we describe the identification, cloning, and characterization of TFIIIB50, a novel homologue of TFIIB and TFIIIB90. TFIIIB50 and tightly associated factors, along with TBP and TFIIIB150, reconstitute human TFIIIB-alpha activity. Thus, higher eukaryotes, in contrast to the yeast Saccharomyces cerevisiae, have evolved two distinct TFIIB-related factors that mediate promoter selectivity by RNA polymerase III.
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PMID:A stable complex of a novel transcription factor IIB- related factor, human TFIIIB50, and associated proteins mediate selective transcription by RNA polymerase III of genes with upstream promoter elements. 1112 Oct 26

The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (tau(131)) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37 degrees C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5' ends. Maturation of tRNA was found to be aberrant in bdp1-Delta 253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1 Delta 253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.
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PMID:Essential roles of Bdp1, a subunit of RNA polymerase III initiation factor TFIIIB, in transcription and tRNA processing. 1197 60

RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNA(Leu) genes in cells decreases when ERK is inactivated. Furthermore, mutation of an ERK docking domain or phosphoacceptor site in BRF1 prevents serum induction of pol III transcription. These data identify a novel target for ERK, and suggest that its ability to stimulate biosynthetic capacity and growth involves direct transcriptional activation of tRNA and 5S rRNA genes.
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PMID:The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB. 1274 36

The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.
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PMID:TATA-binding protein mutants that are lethal in the absence of the Nhp6 high-mobility-group protein. 1522 42

RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.
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PMID:Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors. 2041 82

PROBLEM STATEMENT: RNA polymerase III (RNA pol III) is responsible for transcribing many of the small structural RNA molecules involved in RNA processing and protein translation, thereby regulating the growth rate of a cell. RNA pol III transcribes both gene internal (tRNA) and gene external (U6 snRNA) promoters and proper initiation by RNA polymerase III requires the transcription initiation factor TFIIIB. TFIIIB has been shown to be a target of repression by tumor suppressors such as ARF, p53, RB and the RB-related pocket proteins. Also, TFIIIB activity is stimulated by the oncogenes c-Myc and the ERK mitogen-activated protein kinase. Recently, two TFIIIB subunits, BRF1 and BRF2, have been demonstrated to behave as oncogenes, making deregulation of TFIIIB activity and thus RNA pol III transcription an important step in tumor development. PTEN is a commonly mutated tumor suppressor regulating cell growth, proliferation and survival. Thus, we sought to examine the potential role of PTEN in regulating U6 snRNA transcription. APPROACH: We examined the potential for PTEN to regulate U6 snRNA transcription using in vitro RNA pol III luciferase assays, western blotting and deletion analysis in cancer cell lines differing in their PTEN status. RESULTS: Using breast, cervical, prostate and glioblastoma cancer cells we demonstrate: (1) PTEN inhibition of gene external RNA pol III transcription is cell type specific, (2) PTEN-mediated inhibition of U6 transcription occurs via the C2 lipid-binding domain and (3) PTEN repression of U6 transcription occurs, at least in part, through the TFIIIB subunit BRF2. CONCLUSION/RECOMMENDATIONS: Our data demonstrates that regulation of the U6 snRNA gene by PTEN is mediated, in part by the TFIIIB oncogene BRF2, potentially identifying novel targets for chemotherapeutic drug design.
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PMID:Inhibition of U6 snRNA Transcription by PTEN. 2147 60


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