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)

Cell-free extracts from Xenopus laevis oocytes (ovarian tissue), unfertilized eggs, embryos, and cultured kidney cells direct accurate transcription of cloned 5 S RNA and tRNA genes. Fractionation of these extracts by conventional ion exchange chromatography shows that multiple components are essential for the transcription of each of these genes by RNA polymerase III. Two chromatographically distinct fractions from ovary, egg, embryo, and kidney cell extracts are necessary and sufficient, in the presence of purified RNA polymerase III, for the transcription of tRNA genes. Transcription of 5 S RNA genes requires components present in these same fractions as well as the previously described 5 S gene-specific factor (TFIIIA). The analogous chromatographic fractions from different tissue extracts are functionally interchangeable, consistent with the suggestions that the endogenous factors which are necessary and sufficient for purified gene transcription are general (and not tissue-specific) factors.
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PMID:Multiple factors involved in the transcription of class III genes in Xenopus laevis. 713 Jan 90

We find striking similarities in promoter structure and requirements for template commitment on 5S RNA and tRNA genes from silkworms. The promoters are nearly the same size (approximately 160 bp) and include flanking as well as internal sequences. To analyze the factor requirements for 5S RNA transcription complex assembly in a completely homologous system, we have isolated a silkworm fraction that is highly enriched for the 5S RNA-specific transcription factor, TFIIIA. Using this fraction, together with the other silkworm fractions, TFIIIB, TFIIIC, TFIIID and RNA polymerase III, we demonstrate that the requirements for 5S RNA transcription complex assembly are very similar to those previously established for a tRNA(C)(Ala) gene. Specifically, no individual factor fraction is sufficient for commitment of silkworm 5S RNA genes to transcription complex assembly. Rather, combinations of at least three factor fractions are required. Our observation that more than one subset of factors is competent for commitment suggests that silkworm 5S RNA genes further resemble tRNA(C)(Ala) genes in their ability to use multiple pathways for transcription complex formation.
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PMID:Silkworm TFIIIA requires additional class III factors for commitment to transcription complex assembly on a 5S RNA gene. 773 3

Transcription of large rRNA precursor and 5S RNA were examined during encystment of Acanthamoeba castellanii. Both transcription units are down regulated almost coordinately during this process, though 5S RNA transcription is not as completely shut down as rRNA transcription. The protein components necessary for transcription of 5S RNA and tRNA were determined, and fractions containing transcription factors comparable to TFIIIA, TFIIIB, and TFIIIC, as well as RNA polymerase III and a 3'-end processing activity, were identified. Regulation of 5S RNA transcription could be recapitulated in vitro, and the activities of the required components were compared. In contrast to regulation of precursor rRNA, there is no apparent change during encystment in the activity of the polymerase dedicated to 5S RNA expression. Similarly, the transcriptional and promoter-binding activities of TFIIIC are not altered in parallel with 5S RNA regulation. TFIIIB transcriptional activity is unaltered in encysting cells. In contrast, both the transcriptional and DNA-binding activities of TFIIIA are strongly reduced in nuclear extracts from transcriptionally inactive cells. These results were analyzed in terms of mechanisms for coordinate regulation of rRNA and 5S RNA expression.
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PMID:Coordinate regulation of ribosomal component synthesis in Acanthamoeba castellanii: 5S RNA transcription is down regulated during encystment by alteration of TFIIIA activity. 776 Aug 28

Xenopus TFIIIA gene expression is developmentally regulated. The per cell level of steady-state TFIIIA mRNA present in oocytes is drastically reduced (10(6)-fold) in adult somatic cells, suggesting regulation at the transcriptional level. This is supported by the presence of TFIIIA mRNAs with different 5'-ends in oocytes and in somatic cells, which suggests the presence of distinct cell-/stage-specific promoters, i.e. a strong oocyte-specific and a weak somatic-specific promoter. Here, by mapping the 5'-ends of TFIIIA RNAs found in Xenopus somatic cells, we document the activity not only of an upstream somatic-specific promoter, but also of a down-stream "oocyte-specific" promoter that gives rise to a minor population of somatic TFIIIA mRNAs initiated from the same transcription start site utilized in oocytes. This result explains the presence of an oocyte-type form of TFIIIA protein in somatic cells. Furthermore, by characterizing transcription from the Xenopus TFIIIA promoter in somatic cell nuclear extracts and comparing the in vitro transcription start sites with the 5'-ends of endogenous somatic TFIIIA transcripts in poly(A)+ and poly(A)- RNA fractions, we show that RNA polymerase III initiates at position -70 of the "oocyte-specific" core promoter and transcribes through the promoter and into the TFIIIA coding sequences in somatic cells. These results suggest a possible polymerase III transcription-mediated down-regulation of the oocyte-specific TFIIIA promoter in Xenopus somatic cells.
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PMID:Overlapping transcription by RNA polymerases II and III of the Xenopus TFIIIA gene in somatic cells. 792 74

We report the characterization of a mutation affecting tau 138, the largest subunit of yeast transcription factor IIIC (TFIIIC). A previously described thermosensitive mutation (tsv115), tightly linked to the centromere of chromosome I (Harris, S.D., and Pringle, J.R. (1991) Genetics 127, 279-285) is shown to lie in the TFC3 gene which encodes tau 138. The tau 138 subunit carrying this mutation bears a single substitution of Glu for Gly at position 349 (G349E). In extracts from mutant cells, both the level of TFIIIC and its affinity for tDNA were found to be reduced. The tDNA binding activity of mutant TFIIIC protein was very sensitive to mild heat treatments, and TFIIIC-DNA interaction was inhibited at moderate salt concentrations, as evidenced by gel shift assays. In addition, the tsv115 mutation affected 5 S RNA synthesis in vitro, suggesting that the tau 138 subunit also plays a role in recognition of the TFIIIA-5 S DNA complex. Multicopy suppressors of the TFIIIC defect were sought to reveal components participating in TFIIIC function. One class of suppressors encodes known components of the transcription machinery: two TFIIIC subunits, tau 95 and tau 131, the 70-kDa subunit of TFIIIB, TBP, and a shared subunit of RNA polymerase (pol) I, II, and III, ABC10 alpha; it also includes genes potentially related to pol III function, such as SRP40 which also suppresses a mutation in a subunit shared by RNA polymerases I and III. A second class of suppressors is not involved in transcription but alleviates the main physiological defects of mutant cells. It includes RPR1 and NOP1, required for the maturation of pre-tRNA and pre-rRNA, respectively.
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PMID:A mutation in the largest subunit of yeast TFIIIC affects tRNA and 5 S RNA synthesis. Identification of two classes of suppressors. 808 43

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.
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PMID:High level expression in E. coli and purification of yeast transcription factor IIIA. 809 41

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.
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PMID:Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC. 816 61

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.
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PMID:A position-dependent transcription-activating domain in TFIIIA. 824 67

We have described elsewhere a number of the properties of a set of mutant forms of Xenopus transcription factor IIIA (TFIIIA) containing single amino acid substitutions that result in the structural disruption of individual zinc finger domains. These "broken finger" proteins have now been analyzed with respect to their ability to support transcription of 5S rRNA genes in vitro. Disruption of any one of the first six zinc fingers of TFIIIA has no discernible effect on the activity of the protein in supporting 5S rRNA synthesis in standard in vitro transcription assays, despite the fact that some of these mutant proteins exhibit large decreases in their binding affinity for 5S rRNA genes in binary complexes. These results indicate that the activity of TFIIIA as a transcription factor can be largely independent of its equilibrium binding constant for the 5S rRNA gene in the absence of other components of the RNA polymerase III transcriptional apparatus. In fact, this finding is consistent with the known pathway and kinetics of assembly of 5S rRNA transcription complexes. In contrast to the results obtained with finger 1-6 mutants, analogous mutations in zinc fingers 7-9 of TFIIIA result in moderate to complete loss of transcriptional activity. We interpret these results to mean that the three C-terminal zinc fingers of TFIIIA are not only involved in binding to the internal control region of 5S rRNA genes but are also required, either directly or indirectly, for higher-order interactions that are important in transcription complex assembly, stability, or activity.
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PMID:The role of zinc fingers in transcriptional activation by transcription factor IIIA. 841 19

In eukaryotes 5S rRNA genes are transcribed by RNA polymerase III. These genes occur in D. discoideum on the ca. 90 copies of an extrachromosomal palindrom together with the other ribosomal RNAs, which are generally transcribed by RNA polymerase I. A 5S rRNA gene has been isolated and its nucleotide sequence as well as the organization of the gene relative to the RNA polymerase I operon has been determined. The sequence of the coding region corresponds exactly to an earlier published 5S rRNA sequence. The genes are located just downstream from the 26S RNA and transcription orientations of the pol I genes and the pol III gene point into the same direction, away from the centromer of the palindrom. The isolated gene appears to be functional since it serves as a specific target for a nuclear protein, most likely TFIIIA. A genomic copy of a 5S rRNA pseudogene has been isolated from the D. discoideum strain V12. This pseudocopy contains nucleotides 52 to 82 of a bona fide 5S rRNA gene with only three mismatches. It resides 78 nucleotides downstream from the glu13(UUC) tRNA gene which in the D. discoideum strain V12 is associated with the retrotransposable element DRE.
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PMID:The Dictyostelium discoideum 5S rDNA is organized in the same transcriptional orientation as the other rDNAs. 846 Oct 13


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