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)

The S. cerevisiae RNA polymerase III (pol III) transcription factor TFIIIB binds to DNA upstream of the transcription start site of the SUP4 tRNA(Tyr) gene in a TFIIIC-dependent reaction and to the major 5S rRNA gene in a reaction requiring TFIIIC and TFIIIA. It is shown here that TFIIIB alone correctly positions pol III for repeated cycles of transcription on both genes, with the same efficiency as fully assembled transcription complexes. Thus, TFIIIB is the sole transcription initiation factor of S. cerevisiae pol III; TFIIIC and TFIIIA are assembly factors for TFIIIB. The TFIIIB-dependent binding of pol III to the SUP4 tRNA and 5S rRNA genes has been analyzed in binary (protein and DNA only) and precisely arrested ternary (protein, DNA, and RNA) transcription complexes. Pol III unwinds at least 14 bp of DNA at the SUP4 transcription start in a temperature-dependent process. The unwound DNA segment moves downstream with nascent RNA as a transcription bubble of approximately the same size.
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PMID:S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors. 240 11

B2 genes are rodent-specific middle repetitive elements transcribed by RNA polymerase III. They are expressed in the ectoderm and mesoderm but not in the embryonic or extraembryonic endoderm of early mouse embryos. This tissue specificity is mimicked in vitro by embryonal carcinoma and embryonic stem cell lines. Nuclear run-on experiments show that the down-regulation of B2 genes during F9 embryonal carcinoma cell differentiation into endoderm occurs at the transcriptional level and that other class III genes, including those encoding tRNA, show a similar response. We have used cell-free extracts to investigate the molecular mechanisms responsible. The specific down-regulation of transcription by RNA polymerase III during F9 cell differentiation is due to a reduction in the activity of the general class III transcription factor TFIIIB.
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PMID:Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation. 259 61

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.
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PMID:Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes. 266 66

We have used an Eppendorf centrifuge for isolation of transcription complexes assembled on VARNA genes and other related genes with NTP-depleted cell-free extracts. Similar to the 5 S rRNA gene, sedimentable, stable transcription preinitiation complexes could be assembled from two VARNA genes, two EB virus-specific EBER genes, four human tRNA genes, and one human Alu-family RNA gene, suggesting that the 5 S rRNA-specific transcription factor, TFIIIA, was not required for formation of these sedimentable, stable preinitiation complexes. Parameters affecting assembly of these complexes were sequences in circular DNA templates, sizes and sequences of linear DNA templates, temperature and incubation time. These complexes were stable at from 4 to 37 degrees C, and somewhat stable to salt wash. From results of effects of various mutations on assembly of these sedimentable complexes, we concluded that they were transcription machineries. Addition of the supernatant and partially purified factors to salt-washed complexes stimulated their transcription, we concluded that these sedimentable complexes were minimal transcription machineries containing suboptimal quantities of loosely bound transcription factors, TFIIIB, and RNA polymerase III. DNase 1 footprints of these sedimentable preinitiation complexes showed that two regions were protected, from +34 to +80 including the B block promoter element, and from +98 to +105. Similar DNase 1 footprints were also obtained from salt-washed complexes and stable preinitiation complexes isolated by molecular sieve column chromatography.
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PMID:Formation of large, sedimentable transcription complexes with VARNA genes and other related genes. 272 76

Transcription factor IIIC2 (TFIIIC2), together with other transcription factors (TFIIIB and TFIIIC1), is required for the in vitro transcription of tRNA and adenovirus VA genes by RNA polymerase III. Previous studies have shown that TFIIIC2 is a high molecular weight (approximately 500,000) protein which binds with high affinity to the B-box promoter element of tRNA-type genes. A polypeptide of Mr approximately 250,000 is in close association with DNA in the specific complex between TFIIIC2 and the B-box promoter element. Here we describe the purification of TFIIIC2 by a factor of approximately 25,000 from nuclear extracts of HeLa cells by ionic exchange, affinity, and hydrophobic chromatography and sedimentation velocity centrifugation. The most purified fractions contain polypeptides of approximately 230 kDa (corresponding to the polypeptide which can be cross-linked to VA1 DNA), 110, 100, 80, and 60 kDa which co-sediment with TFIIIC2 B-box specific binding and in vitro transcriptional activities.
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PMID:Purification and characterization of transcription factor IIIC2. 273 44

Human transcription factor IIIC (TFIIIC) is an initiation factor required for the in vitro transcription of 5 S RNA, tRNA, and adenovirus viral-associated (VA) RNA genes by RNA polymerase III. A TFIIIC activity which complemented purified TFIIIB and RNA polymerase III fractions for VA transcription was highly purified from cultured HeLa cells. This activity copurified through all chromatographic procedures, including B-block oligodeoxynucleotide affinity chromatography, with the two forms of TFIIIC detected by gel mobility shift assays with the VA gene (Hoeffler, W.K., Kovelman, R., and Roeder, R.G. (1988) Cell 53, 907-920). Both specific binding activity to the VAI gene and TFIIIC transcription activity were inhibited by the alkylating agents diisopropyl fluorophosphate, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and N-ethylmaleimide, and to a lesser extent by N alpha-p-tosyl-L-lysine chloromethyl ketone, whereas neither activity was inhibited by phenylmethylsulfonyl fluoride. These data suggest further that the DNA binding and transcription assays scored the same protein(s). TPCK and N-ethylmaleimide inactivated TFIIIC solely through thiol group modification, since prior modification with the reversible thiol reagent 2,2'-dithiopyridine prevented permanent inactivation. The involvement of reduced thiol groups in the specific binding of TFIIIC to the VAI gene was further indicated by an increase in TFIIIC binding activity upon addition of dithiothreitol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mr = 126,000 polypeptide both eluted from a B-block oligodeoxynucleotide affinity column with the DNA binding and transcription activities of TFIIIC and was specifically cross-linked by UV to a 5-bromo-2-deoxynucleotide-substituted B-block oligodeoxynucleotide. The near identity of the TFIIIC molecular weight determined by gel filtration on SOTA Phase GF 200 (Mr = 140,000) suggests that TFIIIC in solution (in the presence of 0.3 M NaCl at pH 7.0) consists of a single polypeptide which is fairly globular in nature.
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PMID:Human transcription factor IIIC (TFIIIC). Purification, polypeptide structure, and the involvement of thiol groups in specific DNA binding. 280 67

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.
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PMID:Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors. 282 18

Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter.
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PMID:Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells. 335 99

tRNA genes and adenovirus viral-associated (VA) genes are transcribed by RNA polymerase III. Transcription of these genes in vitro requires two protein fractions containing transcription factors designated TFIIIB and TFIIIC, in addition to RNA polymerase III. We report that the TFIIIC fraction derived from human cells in culture can be separated into two functional components, which we call TFIIIC1 and TFIIIC2. Both TFIIIC1 and TFIIIC2 fractions are required for in vitro transcription of the VA1 gene. In DNase I "footprinting" experiments, the TFIIIC2 fraction protects the internal control region termed the B block. Addition of the TFIIIC1 fraction extends the footprint over the internal control region called the A block. TFIIIC1 activity is the limiting transcription factor activity required for VA1 transcription in the crude extract. TFIIIC2 activity sediments as a large component of approximately 18 S, while TFIIIC1 activity sediments at approximately 9 S. These data indicate that the two activities are unique components and when added together reconstitute TFIIIC activity.
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PMID:Resolution of human transcription factor TFIIIC into two functional components. 347 69

Novobiocin concentrations normally used to inhibit a putative eukaryotic DNA gyrase have been found to inhibit transcription of a yeast 5S rRNA gene using an in vitro yeast transcription system. Purified RNA polymerase III and three yeast transcription factors (chromatographically separated, partially purified and free of any detectable gyrase activity) were used. Novobiocin prevents specific transcription if added to the in vitro system immediately prior to the addition of transcription factors and RNA polymerase. If a stable transcription factor complex is allowed to form prior to the addition of novobiocin, concentrations of novobiocin as high as 1000 micrograms/ml have no effect on in vitro transcription. Transcription factors TFIIIA and TFIIIC are able to be stably sequestered onto 5SrDNA-cellulose, but factor TFIIIB is not able to associate with the 5SrDNA-TFIIIA-TFIIIC complex in the presence of novobiocin. Although novobiocin is able to precipitate other basic proteins, it does not appear to precipitate any of these class III gene transcription factors, but instead appears to act by disrupting specific factor-factor interactions.
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PMID:Novobiocin inhibits interactions required for yeast TFIIIB sequestration during stable transcription complex formation in vitro. 354 36


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