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)

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.
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PMID:Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains. 762 63

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

The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF.
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PMID:Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III. 881 54

Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 was expressed in Escherichia coli and purified to homogeneity. The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor. However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA. The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex. Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 +/- 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP.TFIIIB70.DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm. A Kd of 44 +/- 23 nM characterizes the TFIIIB70 concentration dependence of the TBP.TFIIIB70 cooperativity. The relationship deltalog K/deltalog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.
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PMID:Expression and purification of the RNA polymerase III transcription specificity factor IIIB70 from Saccharomyces cerevisiae and its cooperative binding with TATA-binding protein. 895 1

TFIIIB, the initiation factor for transcription by RNA polymerase III (pol III) is, in yeast, composed of three subunits: TBP, TFIIIB70/Brf1 and TFIIIB90. To determine the extent to which each of these subunits is limiting for pol III transcription, the effect of overexpressing each subunit was assessed on the expression of wild-type and promoter mutant pol III genes both in vivo and in vitro . In vivo , we find that the synthesis of wild-type pol III genes is not limited to a significant extent by the level of any TFIIIB subunit. There is, however, a two-fold increase in the synthesis of the promoter mutant gene, sup9-e A19-supS1 , in strains overexpressing TFIIIB70. The findings suggest that overexpression of TFIIIB70has a differential effect on the expression of pol III genes with strong versus weak promoters. In vitro transcription assays support this conclusion and reveal an inverse correlation between the transcriptional response to TFIIIB70overexpression and promoter strength. The individual TFIIIB subunits are nuclear by immunofluorescence and are calculated to have nuclear concentrations in the low micromolar range. In comparison, the factors are diluted 100-fold or more in whole cell extracts. This dilution accounts for the generally limiting nature of TFIIIB70in pol III gene transcription in vitro.
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PMID:A differential response of wild type and mutant promoters to TFIIIB70 overexpression in vivo and in vitro. 958 Jun 84

The yeast TFIIIB transcription factor is composed of three components, TBP, TFIIIB90 or B", and TFIIIB70 or BRF. TFIIIB70 is a pivotal component since it interacts with TBP, TFIIIC and RNA polymerase III (pol III). In order to better understand the role of TFIIIB70, we mutagenized extensively three evolutionary conserved motifs of its pol III-specific C-terminal extension. Conditional mutations lying in conserved regions II and III were obtained, some of which altered the interaction with the C34 subunit of pol III and were co-lethal with rpc34 mutations. Two conditional mutations in region II impaired the interaction with TBP and were suppressed by its overexpression. The pattern of suppression of the strongest mutation by overexpression of various mutant TBP, suggested a contact between TBP-R220 and TFIIIB70-D464 residues in vivo. As expected, this TFIIIB70 mutation impaired the assembly of TFIIIB. TFIIIC.DNA complexes and affected in vitro transcription of the SUP4 tRNA gene. Our results underscore the important role of region II of TFIIIB70 in pre-initiation as well as transcription complex assembly via C34 and TBP binding.
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PMID:Mutagenesis of yeast TFIIIB70 reveals C-terminal residues critical for interaction with TBP and C34. 1032 59

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

High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation.
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PMID:Kinetic trapping of DNA by transcription factor IIIB. 1148 28

The TFIIIB transcription factor is the central component of the RNA polymerase III transcriptional machinery. In yeast, this factor is composed of three essential polypeptides TBP, TFIIIB70 and TFIIIB90, that are sufficient as recombinant proteins, together with TFIIIC, to promote accurate transcription in vitro. Here we show that a partially purified fraction, named B", that contains the TFIIIB90 subunit, displays properties distinct from recombinant TFIIIB90. This fraction contains at least a component that interacts with DNA*TFIIIC complexes, either alone or in combination with TFIIIB90, and increases the resistance of the complexes to heparin treatment. In addition, primer extension and single round transcriptions experiment reveal a different start-site selection pattern directed by B" or rTFIIIB90. In mixing experiments, we show that an activity in B", distinct from TFIIIB90, can promote transcription initiation at the +1 site without affecting the rate of preinitiation complex formation. Our data suggest the existence of at least one new component that participates in preinitiation complex formation and influences start-site selection by RNA polymerase III.
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PMID:B"-associated factor(s) involved in RNA polymerase III preinitiation complex formation and start-site selection. 1158 9

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