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)

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.
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PMID:p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner. 894 63

The structure of the Saccharomyces cerevisiae RNA polymerase III transcription complex on the SUP4 tRNATyr gene was probed at distances of approximately 10 to approximately 23 A from the C-5 methyl of thymidine in the major groove of DNA using photoreactive aryl azides attached to deoxyuridine by variable chain lengths. The nucleotide analogs contained an azidobenzoyl group attached with chain lengths that were incrementally increased by approximately 4. 3 A by inserting 1-3 glycine residues into the chain. Another photoreactive deoxyuridine analog was made that contained a butyl chain (ABU-dUMP) to assess the effect of the chain's hydrophobicity on its ability to photoaffinity label the transcription complex. These nucleotide analogs were incorporated at base pairs (bp) -26/-21, -17, or -3/-2 on the nontranscribed strand of the SUP4 tRNATyr gene along with an [alpha-32P]dNMP by primer extension using an immobilized single-stranded DNA template annealed to specific oligonucleotides. The 27-kDa subunit of TFIIIB or the TATA box binding protein was photoaffinity labeled at bp -26/-21 with nucleotide analogs containing a approximately 19- or approximately 23-A chain and not with shorter chains of approximately 10 to approximately 15 A in length. The B" subunit of TFIIIB (Mr = 90 kDa) was photoaffinity labeled at bps -26/-21 with DNA containing a approximately 14-A chain and not with shorter or longer chains. Cross-linking of the B" subunit was inhibited by binding of RNA polymerase III (Pol III) to the TFIIIB-DNA complex and suggested that Pol III binding causes a conformational change in the TFIIIB-DNA complex resulting in the displacement of the 90-kDa subunit at bps -26/-21. Next, the chain length dependence of photoaffinity labeling the 34-kDa subunit of Pol III at bps -17 and -3/-2 indicated that the 34-kDa subunit of Pol III is slightly removed from the major groove at bp -17 in the initiation complex and makes closer contact at bps -3/-2 in a stalled elongation complex.
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PMID:Mapping the contacts of yeast TFIIIB and RNA polymerase III at various distances from the major groove of DNA by DNA photoaffinity labeling. 895 50

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Using Xenopus egg extracts shifted to the mitotic state with recombinant cyclin B1 protein, we have been able to reproduce mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts in the absence of added cyclin, but is strongly repressed by the induction of cdc2/cyclin B (maturation/mitosis promoting factor, MPF) kinase activity in the mitotic extract. Studies with protein kinase inhibitors show that protein phosphorylation is required for repression. Add-back experiments indicate that repression of class III gene transcription is due to inactivation of the transcription factor TFIIIB. TFIIIB is composed of the TATA-box binding protein (TBP) and TBP-associated factors of 75 and 92 kDa. In the present study, we show that TBP and a polypeptide of 92 kDa are substrates of the mitotic kinase in highly purified TF- IIIB fractions. We also show that a phosphatase present in the Xenopus egg extract can reactivate transcription after repression by the mitotic kinases. This result suggests a mechanism for reactivation of transcription after exit from mitosis into the G1 phase of the cell cycle. As for pol III genes, purified cdc2/cyclin B kinase is sufficient to inhibit transcription by RNA polymerase II in a reconstituted transcription system containing the basal transcription factors and polymerase.
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PMID:Repression of RNA polymerase II and III transcription during M phase of the cell cycle. 898 11

Ty3, a gypsylike retrotransposon of budding yeast, integrates at the transcription initiation site of genes transcribed by RNA polymerase III (pol III). It was previously shown that integration in vitro requires intact promoter elements and the pol III transcription factors TFIIIB and TFIIIC. In order to test the effect of pol III on integration, increasing amounts of a pol III-containing fraction were added to Ty3 in vitro integration reactions. The pol III-containing fraction was inhibitory to integration. These results are consistent with a model where the Ty3 integration complex and pol III recognize similar features of the stable transcription complex and compete with each other for access to the transcription initiation site.
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PMID:RNA polymerase III interferes with Ty3 integration. 910 9

The tumour suppressor protein RB restricts cellular growth. This may involve inhibiting the synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III. We have shown previously that RB can repress pol III transcription when overexpressed either in vitro or in vivo. We also demonstrated that pol III activity is elevated substantially in primary fibroblasts from RB-deficient mice. Here we address the molecular mechanism of this regulation. RB is shown to repress all types of pol III promoter. It can do this even if added after transcription complex assembly. Functional assays demonstrate that RB targets specifically the general pol III factor TFIIIB. A physical interaction between TFIIIB and RB is indicated by fractionation, pull-down and immunoprecipitation data. We show that TFIIIB activity is elevated in primary fibroblasts from RB-deficient mice. TFIIIB is a multisubunit complex that includes the TATA-binding protein (TBP) and a TFIIB-related factor called BRF. We show that RB itself contains regions of homology to both TBP and BRF and propose a model in which RB disrupts TFIIIB by mimicking these two components.
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PMID:Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein. 915 32

The retinoblastoma susceptibility gene product (Rb) generally represses RNA polymerase III (Pol III)-directed transcription. This implies that Rb interacts with essential transcription factors. Mutations in either the A or B subdomains in the Rb pocket interfere with Rb-mediated repression of Pol III-directed transcription, which indicates that both subdomains are directly involved in this activity. Addition of either purified TFIIIB or purified TFIIIC2 partially relieves Rb-mediated repression and restores activity to nuclear extracts that had been depleted of essential factors by binding to Rb. Pull down and coimmunoprecipitation experiments as well as functional assays indicate that Rb interacts with both TFIIIB and TFIIIC2 and that the A subdomain is primarily required for binding TFIIIB and the B subdomain for binding TFIIIC2. While Rb interacts with both factors, the A subdomain is more important than the B subdomain in directing Rb-mediated repression, and TFIIIB is the principal target of that activity.
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PMID:RNA polymerase III transcription repressed by Rb through its interactions with TFIIIB and TFIIIC2. 916 41

Transcription by RNA polymerase III involves recruitment of the polymerase by template-bound accessory factors, followed by initiation, elongation, and termination steps. An immunopurification approach has been used to demonstrate that human RNA Pol III is composed of 16 subunits, some of which are apparently modified in HeLa cells. Partial denaturing conditions and sucrose gradient sedimentation at high salt result in the dissociation of a subcomplex that includes hRPC32, hRPC39, and hRPC62. Cognate cDNAs were isolated and shown to encode three subunits that are specific to RNA Pol III and homologous to three yeast subunits. The human RNA Pol III core lacking the subcomplex functions in transcription elongation and termination following nonspecific initiation on a tailed template, but fails to show promoter-dependent transcription initiation in conjunction with accessory factors. The capability for specific transcription initiation can be restored either by the natural subcomplex or by a stable subcomplex composed of recombinant hRPC32, hRPC39, and hRPC62 polypeptides. One component (hRPC39) of this subcomplex interacts physically with both hTBP and hTFIIIB90, two subunits of human RNA Pol III transcription initiation factor IIIB. These data strongly suggest that the hRPC32-hRPC39-hRPC62 subcomplex directs RNA Pol III binding to the TFIIIB-DNA complex via the interactions between TFIIIB and hRPC39.
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PMID:Three human RNA polymerase III-specific subunits form a subcomplex with a selective function in specific transcription initiation. 917 75

HeLa cell nuclear extracts were used to study the mechanism of activation of RNA polymerase II-mediated transcription by the N-terminal transactivation domain (tau1) of the glucocorticoid receptor in vitro. When fused to the Gal4 DNA-binding domain, the tau1 domain activated transcription approximately 9-fold in HeLa nuclear extracts. Using heat treatment to inactivate transcription factor IID (TFIID) in the extract, it was shown that the addition of purified TFIID complex, but not the TATA-binding protein alone, was sufficient to restore this level of activation. The tau1 domain was shown to interact directly with the TFIID complex. This interaction was markedly reduced by a mutation in the tau1 domain that reduces its activity. Furthermore, the interaction was specific for the TFIID complex, since no interaction was seen with TFIIIB, an analogous protein complex involved in RNA polymerase III transcription. The tau1 domain was further shown to interact with the TATA-binding protein subunit of the TFIID complex. These results suggest a mechanism by which the GR tau1 domain might contribute to gene activation by recruitment of the TFIID complex to target promoters.
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PMID:Involvement of the transcription factor IID protein complex in gene activation by the N-terminal transactivation domain of the glucocorticoid receptor in vitro. 928 62

In eukaryotes, TFIIIB is required for proper initiation by RNA polymerase III. In the yeast Saccharomyces cerevisiae a single form of TFIIIB (gammaTFIIIB) is sufficient for transcription of all pol III genes, whereas in extracts derived from human cells two different hTFIIIB complexes exist which we have previously designated as hTFIIIB-alpha and hTFIIIB-beta. Human TFIIIB-alpha is a TBP-free entity and must be complemented by TBP for transcription of pol III genes driven by gene external promoters, whereas hTFIIIB-beta is a TBP-TAF complex which governs transcription from internal pol III promoters. We show that hTFIIIB-beta cannot be replaced by yeast TFIIIB for transcription of tRNA genes, but that the B" component of gammaTFIIIB can substitute for hTFIIIB-alpha activity in transcription of the human U6 gene. Moreover, hTFIIIB-alpha can be chromatographically divided into activities which are functionally related to gammaTFIIIE and recombinant yB"90, suggesting that hTFIIIB-alpha is a human homolog of yeast TFIIIB". In addition, we show that yeast TBP can only be exchanged against human TBP for in vitro transcription of the human and yeast U6 gene but virtually not for that of the yeast tRNA4Sup gene. This deficiency can be counteracted by a mutant of human TBP (R231K) which is able to replace yeast TBP for transcription of yeast tRNA genes in vitro.
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PMID:Functional interchangeability of TFIIIB components from yeast and human cells in vitro. 930 15

Transcription by RNA polymerase III (Pol III) requires multiple general initiation factors that, in isolated form, assemble onto the promoter in an ordered fashion. Here, it is shown that all components required for transcription of the VA1 and tRNA genes, including TFIIIB, TFIIIC, and RNA Pol III, can be coimmunopurified from a HeLa cell line that constantly expresses a FLAG epitope-tagged subunit of human RNA Pol III. This finding of an RNA Pol III "holoenzyme" suggests similarities between transcription initiation by RNA Pol II and RNA Pol III and has led to the identification of a novel general initiation factor (TDF, translation dependent factor) that is present within the holoenzyme. TDF is selectively inactivated during protein synthesis inhibition by cycloheximide and at a late stage of adenovirus infection, thus accounting for the loss of RNA Pol III-mediated transcription of the tRNA and VA RNA genes under these conditions. On the basis of these observations, possible mechanisms for the global regulation of transcription by RNA Pol III and for disassembly of RNA Pol III initiation complexes are proposed.
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PMID:Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition. 930 65


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