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)

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.
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PMID:RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest. 140 24

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.
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PMID:RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae. 140 32

Chloroplast tRNA(Glu) is a bifunctional molecule involved in both the early steps of chlorophyll synthesis and chloroplast protein biosynthesis. Recently the enzymes involved in these processes have been characterized from the green alga Chlamydomonas reinhardtii. In order to investigate whether transcription of the gene for the tRNA(Glu) cofactor would be a possible point of regulation for the biosynthesis of chlorophyll, a homologous in vitro transcription system for C. reinhardtii chloroplast RNA polymerase was developed. The enzymatic activity was partially purified by ion-exchange chromatography to separate it from nuclear RNA polymerases. The highest rate of synthesis was found at pH 7.9, 40 mM KCl, 9 mM MgCl2 and with 25 micrograms plasmid DNA containing the chloroplast tRNA gene per milliliter. The activity was not sensitive to high amounts of alpha-amanitin (500 micrograms/ml) and rifampicin, but was clearly inhibited by heparin. This system was used to undertake a promoter analysis of one of the two identical tRNA(Glu) gene copies found in the C. reinhardtii chloroplast genome (trnE1). The analyzed tRNA gene behaved like a single transcription unit driven by its own promoter. The transcript terminated in a run of four consecutive T residues downstream of the gene. The nucleotide sequence in the 5' region of the gene revealed several potential promoter elements with homology to known chloroplast promoters of the "-10 and -35 region" and the "Euglena promoter" types. Surprisingly, deletion of the complete 5' region did not affect in vitro transcription, while partial deletions of the 5' and 3' coding region totally abolished transcription. This indicates the presence of an internal control region previously found for genes transcribed by nuclear RNA polymerase III. Protein binding studies with the coding region of trnE1 using gel retardation assays demonstrated the formation of two differently sized complexes. In vitro transcription of the tRNA(Glu) gene in extracts prepared from light and dark grown algae failed to demonstrate any significant influence of light on the transcription reaction.
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PMID:Expression of the Chlamydomonas reinhardtii chloroplast tRNA(Glu) gene in a homologous in vitro transcription system is independent of upstream promoter elements. 141 80

A dominant mutation in the PCF4 gene of S. cerevisiae was isolated as a suppressor of a tRNA gene A block promoter mutation. In vitro studies indicate that PCF4 is a stoichiometrically-required RNA polymerase III (pol III) transcription initiation factor. We show that the PCF4-1 mutation increases the number of transcriptionally competent preinitiation complexes by affecting a limiting activity in yeast cell extracts that is squelched by excess TFIIIC. The PCF4 gene encodes a TFIIB homolog whose size, biochemical, and genetic properties are consistent with those of the 70 kd subunit of TFIIIB. The TFIIB homology of PCF4 suggests a means for determining the polymerase specificity of a gene.
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PMID:PCF4 encodes an RNA polymerase III transcription factor with homology to TFIIB. 142 89

A 5789-nucleotide-long EcoRI fragment from the genome of Thermotoga maritima, identified by cross-hybridization to L11, L1, L10, and L12 ribosomal protein gene sequences from Escherichia coli, was cloned and sequenced. The fragment encodes five tRNAs (tRNA(met1), anticodon complementary to AUG; tRNA(met2), AUG; tRNA(thr), ACA; tRNA(tyr), UAC; tRNA(trp), UGG), the transcription termination-antitermination factor nusG, the four 50 S subunit ribosomal proteins L11, L1, L10, and L12, and the amino-terminal portion of the RNA polymerase beta subunit protein. The five tRNA genes, the nusG gene, and the L11, L1, L10, and L12 ribosomal protein genes form a complex transcription unit. Transcripts appear to be initiated from an upstream promoter, P1, located in front of the tRNA(met1) gene and from three internal promoters: P2 is located immediately in front of the tRNA(met2) gene; PL10 is near the beginning of the L1-L10 intergenic space, and PL12 is at the end of the L10 gene sequence. The tRNA sequences are excised from the leader regions of the P1- and P2-initiated transcripts. Three putative but potentially important regulatory sequences were identified within this operon: an L1 translational control site, a transcription attenuator, and a strong rho-independent terminator. The strong terminator located distal to the L12 gene overlaps a fifth promoter, P beta, which is used to initiate transcripts of the downstream RNA polymerase beta subunit gene. The T. maritima NusG protein exhibits 43% amino acid sequence identity when aligned to the E. coli protein; the alignment is interrupted by a large 171-amino acid-long insertion into the T. maritima protein after codon 45.
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PMID:The organization and expression of essential transcription translation component genes in the extremely thermophilic eubacterium Thermotoga maritima. 142 27

RPC53 has previously been shown to encode an essential subunit required for tRNA gene transcription by RNA polymerase C in vivo (Mann, C., Micouin, J.-Y., Chiannilkulchai, N., Treich, I., Buhler, J.-M., and Sentenac, A. (1992) Mol. Cell. Biol. 12, in press). In this paper, we have determined that an unusual rho+ lethality associated with the rpc53::HIS3-1 disruption mutation is due to the inadvertent formation of a Pet56-C53 fusion protein. This fusion protein is missorted to mitochondria, thereby reducing the quantity of the C53 subunit available for RNA polymerase C assembly. We show that the carboxyl-terminal region of C53 contains the essential functional domain of the subunit and that a mutant RNA polymerase containing only this domain is thermolabile for its function in vivo and in vitro. The thermolability of the carboxyl-terminal C53 domain is suppressed by five different genes on multicopy plasmids, including RPC160, encoding the largest subunit of RNA polymerase C and SSD1/SRK1, which has been implicated in the activity of protein phosphatases.
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PMID:Biochemical and genetic dissection of the Saccharomyces cerevisiae RNA polymerase C53 subunit through the analysis of a mitochondrially mis-sorted mutant construct. 142 57

The Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".
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PMID:The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. 145 36

Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.
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PMID:Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. 145 53

We have investigated the requirement for TBP (TATA-binding protein) in transcription mediated by RNA polymerase III (pol III) in fractionated HeLa cell extracts. Two activities, TFIIIB and TFIIIC, found in phosphocellulose fractions PC B and PC C respectively, have been defined as necessary and sufficient, with pol III, for in vitro transcription of tRNA genes. Depletion of TBP from PC B, using antibodies raised against human TBP, is shown to inhibit the pol III transcriptional activity of the fraction. Furthermore, TBP is present in fractions with human TFIIIB activity, and a proportion of TBP cofractionates with TFIIIB over four chromatographic purification steps. TFIIIB fractions are capable of supplying TBP in the form necessary for pol III transcription, and cannot be substituted by fractions containing other TBP complexes or TBP alone. The use of a 5S RNA gene and two tRNA templates supports the general relevance of our findings for pol III gene transcription. Purified TFIIIB activity can also support pol II-mediated transcription, and is found in a complex of approximately 230kD, suggesting that TFIIIB may be the same as the previously characterized B-TFIID complex (1,2). We suggest that transcription by the three RNA polymerases is mediated by distinct TBP-TAF complexes: SL1 and D-TFIID for pol I and pol II respectively, and TFIIIB for pol III.
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PMID:Cofractionation of the TATA-binding protein with the RNA polymerase III transcription factor TFIIIB. 146 21

We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.
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PMID:Pseudouridine in the anticodon G psi A of plant cytoplasmic tRNA(Tyr) is required for UAG and UAA suppression in the TMV-specific context. 146 24


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