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

We have measured the transcription of Xenopus laevis oocyte and somatic-type 5 S RNA genes in S-150 extracts prepared from ovaries of mature X. laevis females (Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Cell 37, 33-41). We find that somatic-type 5 S genes are transcribed at least 40-fold more efficiently than oocyte-type 5 S genes. Since adenovirus VA, Xenopus OAX, and Xenopus tRNAMet genes are all transcribed at levels similar to that of the somatic-type 5 S gene, this difference reflects a low level of oocyte-type 5 S gene transcription. Somatic-type 5 S transcription is a linear function of somatic-type 5 S DNA concentration and this, together with the efficient transcription of other class III genes, suggests that RNA polymerase III and the general class III transcription factors are not limiting under the conditions used here. Moreover, the 5 S gene-specific transcription factor TFIIIA does not limit transcription under these conditions as preincubation with purified TFIIIA does not alter transcription of either gene, and both genes exhibit characteristic TFIIIA footprints under transcription conditions in the S-150. Somatic-type 5 S DNA incubated in the S-150 and then injected into oocyte nuclei is actively transcribed whereas oocyte-type 5 S DNA treated in the same manner is inactive. We conclude that factors in the S-150 distinguish between somatic and oocyte-type 5 S genes, assembling active complexes preferentially on the former and inactive complexes preferentially on the latter. This process is not explained by binding properties of transcription factors for these genes as currently understood and represents a previously unrecognized mechanism of transcriptional regulation of the Xenopus 5 S genes.
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PMID:Differential transcription of Xenopus oocyte and somatic-type 5 S genes in a Xenopus oocyte extract. 244 54

Xenopus transcription factor IIIA (TFIIIA) is expressed in Escherichia coli by utilizing one plasmid with a T7 RNA polymerase gene and another plasmid with TFIIIA cDNA cloned downstream of a T7 promoter. Wild-type TFIIIA and a TFIIIA deletion mutant, isolated from E. coli cell extracts, are identified by antiserum against native TFIIIA purified from Xenopus immature oocytes. DNase I protection experiments indicate that wild-type TFIIIA, synthesized from a full-length TFIIIA cDNA, binds specifically to the coding and noncoding strands of the 5 S RNA gene. The TFIIIA deletion mutant, expressed from TFIIIA cDNA lacking the coding sequence for the N-terminal 29 amino acids, fails to bind specifically to the 5 S RNA gene as judged by its inability to protect to any degree the coding or noncoding strands of the gene from DNase I digestion. Both wild-type TFIIIA and the N-terminal deletion mutant promote DNA renaturation.
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PMID:Deletion of the N-terminal region of Xenopus transcription factor IIIA inhibits specific binding to the 5 S RNA gene. 244 98

Kinetic and titration analyses are used to elucidate the mechanism by which Xenopus transcription factor IIIA (TFIIIA), a protein required for 5 S RNA synthesis by RNA polymerase III, promotes DNA renaturation. TFIIIA promotes 50% renaturation of complementary strands (303 bases) in 45 s. Analyses of the renaturation kinetics indicate the rate-limiting step in this TFIIIA-dependent reaction is first order. TFIIIA-dependent DNA renaturation is a stoichiometric rather than a catalytic process. The renaturation rates for specific and nonspecific DNA are very similar, indicating lack of sequence specificity in this TFIIIA-dependent process. In the nanomolar concentration range of protein and DNA, renaturation occurs at a ratio of about one TFIIIA molecule/single strand (303 bases). Elevated reaction temperatures strongly stimulate TFIIIA-dependent DNA renaturation; at 45 degrees C, renaturation of the 303-base pair fragment nears completion in about 5 s. The ability of TFIIIA to rapidly promote DNA renaturation is unique when compared with Escherichia coli recA protein, single-stranded DNA binding protein, or bacteriophage T4 gene 32 protein. This mechanism by which TFIIIA promotes DNA renaturation is compatible with features of 5 S RNA gene transcription.
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PMID:Xenopus transcription factor IIIA-dependent DNA renaturation. 246 Apr 59

Multiple stages of protein-DNA interaction in the assembly of RNA polymerase III transcription complexes on a Saccharomyces cerevisiae 5S rRNA gene have been distinguished by DNase I "footprinting" and gel retardation. Transcription factor IIIA interacts with approximately 35 base pairs of the internal promoter region. Transcription factors IIIC and IIIB incrementally extend the interaction along the 5S gene, if, and only if, transcription factor IIIA is also bound. Complexes assembled from the complete set of purified transcription factors or from a complete transcription system extend over the entire transcription unit together with almost 50 base pairs of 5' flanking sequence.
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PMID:Multiple states of protein-DNA interaction in the assembly of transcription complexes on Saccharomyces cerevisiae 5S ribosomal RNA genes. 264 82

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E. coli utilizing a bacteriophage T7 RNA polymerase system. TFIIIA or deletion mutant TFIIIAs, isolated from E.coli cell extracts, were identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA purified from Xenopus immature oocytes. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control gene (ICR) of the Xenopus 5S RNA gene from DNase I digestion. Intact protein, synthesized from a full-length TFIIIA cDNA, bound specifically to the entire ICR (+96 to +43) and promoted 5S RNA gene transcription in vitro. One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger (designated from the N-terminus, amino acids 103-132) protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 (deletion of amino acids 200-224) protected the 5S gene ICR from positions +96 to +63. The DNase I protection patterns of these mutant proteins are consistent with the formation of strong ICR contacts by those regions of the protein on the N-terminal side of the mutation but not by those regions on the C-terminal side of the mutation. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR. These internal deletion mutants promoted 5S RNA synthesis in vitro and DNA renaturation.
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PMID:Internal deletion mutants of Xenopus transcription factor IIIA. 269 11

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

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

We have assembled transcriptionally active chromatin on 5S DNA plasmids by using a Xenopus oocyte supernatant and the 5S-specific transcription factor IIIA (TFIIIA). In this system, the 5S RNA gene is accurately transcribed at a rapid rate of 50 transcripts per gene per hr. By following the time course of RNA synthesis during chromatin assembly, the dose response to TFIIIA addition, and the effect of novobiocin on the assembled nucleoprotein, we show that there is a strict correlation between transcriptional activity and the generation of torsionally strained DNA supercoils in "dynamic chromatin." Transcription cannot be the cause of the dynamic structure, because the assembly of this chromatin is unaffected by alpha-amanitin levels that completely block RNA polymerase III. Surprisingly, the dynamic chromatin remains transcriptionally active after relaxation with DNA topoisomerase I, which implies that the essential parameter for chromatin transcription is gyration per se, and not its effect on DNA topology.
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PMID:Gyration is required for 5S RNA transcription from a chromatin template. 300 44

Transcription factor IIIA, which binds to the internal control region of the Xenopus 5S RNA gene has a novel structure consisting of nine tandemly repeated structural units. It was proposed by us that each unit interacts with about 5 bp of DNA. We show here that there is a periodicity on this scale in the DNA sequence and, by fine scale probing with nucleases, a corresponding structural repeat. Similar sequence periodicities are found in the internal control regions of other genes transcribed by RNA polymerase III, and also in the SV40 promoter and a monkey gene region to which the transcription factor Sp1 binds. We propose that transcription factor IIIA is the type of a novel class of transcription factors offering combinatorial possibilities for the specific recognition of DNA.
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PMID:An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. 301 16


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