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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)

High specific activity [beta-32P]ATP and [beta-32P]CTP were used to study in vitro transcriptional initiation and subsequent capping of simian virus 40 (SV40) early and later RNAs. More than 40% of the capped SV40 RNA synthesized in vitro was also polyadenylylated. With [beta-32P]ATP, only adenosine-containing caps were labeled and the incorporated radioactive phosphate was found exclusively in the beta position. Cap digestion patterns showed extensive qualitative and quantitative similarities between these 32P-labeled caps and caps labeled in vivo [Canaani, D., Kahana, C., Mukamel, A. & Groner, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 3078--3082]. With [beta-32P]CTP, only early SV40 RNA was labeled, consistent with the absence of cytosine-containing caps in late transcripts. The [beta-32P]CTP-labeled cap was identified as m7GpppCmpU, which was previously identified as the major cap of in vivo labeled early SV40 mRNA [Kahana, C., Gidoni, D., Canaani, D. & Groner, Y. (1981) J. Virol. 37, 7--16]. This experiment provides biochemical evidence for eukaryotic RNA polymerase II initiation of transcription with CTP. The data imply that, on SV40 DNA, RNA polymerase II initiates transcription at multiple nucleotide sequences and capping occurs at the initiator nucleotide.
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PMID:Specific in vitro initiation of transcription of simian virus 40 early and late genes occurs at the various cap nucleotides including cytidine. 626 66

The oligonucleotides synthesized by purified vesicular stomatitis virus in vitro in the absence of one or more ribonucleoside triphosphate precursors have been studied. The oligonucleotides contained the 5'-terminal sequences of the leader RNA and one or more mRNA's. The promoter-proximal oligonucleotides lacked 5'-terminal cap structure and contained triphosphate A. These results suggest that the RNA polymerase is located at multiple promoter sites on the genome RNA from where it initiates transcription. The capping reaction appears to occur subsequently during RNA chain elongation. We have also demonstrated that a unique dinucleotide, pppGpC, of presently unknown function is synthesized in vitro in large amounts during RNA synthesis or in the presence of GTP and CTP only.
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PMID:Identification of promoter-proximal oligonucleotides and a unique dinucleotide, pppGpC, from in vitro transcription products of vesicular stomatitis virus. 626 24

The in vitro characteristics of human rotavirus transcription have been examined. The virus has an associated RNA polymerase activity which was activated after a heat shock treatment. The enzyme required the presence of the four ribonucleoside triphosphates and a divalent cation (Mg2+), and it required an optimum pH of 8.5. The polymerase was activated by monovalent salts and inhibited by Na PPi. The addition of actinomycin D, alpha-amanitin, or rifampin did not inhibit the polymerase activity. After thermal shock of the virus, at least eight different RNA species were synthesized which may correspond to independent transcripts. Transcription also requires a hydrolyzable form of ATP. Analogs such as beta,gamma-imido ATP or beta,gamma-methylene ATP were inhibitory, whereas others, such as the beta-gamma-imido or methylene analogs of CTP, UTP, or GTP, were not inhibitory. This suggests that ATP is related to reactions other than polymerization, probably to initiation or elongation of RNA molecules, as has been described for vesicular stomatitis virus or vaccinia virus.
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PMID:In vitro transcription catalyzed by heat-treated human rotavirus. 627 Mar 65

Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G. A. (1981) J. Biol. Chem. 256, 8177-8182). We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro. In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts. The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate. In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence. A minor start occurs at residue 142 and uses CTP as the initiating nucleotide. In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively. In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder. Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation. Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.
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PMID:Tandem promoters in the gene for ribosomal protein S20. 630 58

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non-denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re-initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA-binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.
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PMID:Isolation of a transcription complex for ribosomal 5S RNA. 647 47

Termination of transcription at the end of the tryptophan (trp) operon of E. coli at the trp t site is very efficient in vivo, but is only 25% efficient in vitro. To try to resolve this discrepancy, we have altered numerous parameters and report here on the modifications that bring the in vitro results into closer agreement with the in vivo ones. Lowering the concentration of UTP (but not ATP, CTP or GTP) in the transcription mix can greatly improve termination at trp t. With three other terminators structurally similar to trp t, there is no detectable effect of reducing the concentration of any of the four triphosphates. This response at trp t to low UTP is therefore both nucleotide-specific and terminator-specific, suggesting that apparently minor structural differences may still have profound effects upon termination. Increased specificity and sensitivity may also be provided by the NusA protein, which causes RNA polymerase to pause at trp t and at the 1:2 stem of the trp attenuator. NusA protein also enhances termination at trp t, an effect similar to the low UTP response. Further, termination can be slightly improved by including rho factor, resulting in an overall efficiency of almost 100% at trp t.
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PMID:Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli. 675 52

A mutant of Salmonella typhimurium with a defect in the regulation of pyr-gene expression was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5-fluorouracil and 5-fluorouridine. The mutant possesses 4-fold elevated pools of the pyrimidine nucleoside triphosphatases, UTP and CTP. The specific activities of aspartate transcarbamylase and orotate phosphoribosyltransferase are 40-fold and 7-fold higher in the mutant than in the parent strain when grown in minimal media. Furthermore, the synthesis of the two enzymes in the mutant is not repressed following addition of exogenous pyrimidines. The levels of carbamoylphosphate synthase and orotidine 5'-monophosphate decarboxylase are approximately 3-fold enhanced, while the activities of dihydroorotase and dihydroorotate oxidase appear largely unaffected by the mutation. The mutation responsible for these effects was shown to map between two known point mutations in the rpoBC gene cluster encoding the beta and beta' subunits of RNA polymerase. These observations indicate a regulatory function of RNA polymerase in the control of pyr-gene expression in S. typhimurium.
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PMID:RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC. 676 70

The specificity of transcription of Euglena gracilis Z chloroplast DNA by chloroplast DNA-dependent RNA polymerase in a transcriptionally active chromosome (Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045) has been studied. RNA molecules are both initiated and elongated in vitro. The RNA transcripts have been characterized as to their size, nuclease sensitivity, 5'-terminal oligonucleotides, and coding locus on the chloroplast genome. RNA labeled in vitro at the 5' end with [gamma-32P]ATP was digested with RNase T1, RNase A, and S1 nuclease. The resulting 5'-gamma-32P-oligonucleotides were fractionated by gel electrophoresis. In each case, one or two discrete products were obtained, consistent with initiation in vitro only at defined loci. RNA labeled in vitro with [alpha-32P]ATP or CTP has been hybridized to Southern (Southern, E.M. (1975) J. Mol. Biol. 98, 503-517) transfers of restriction endonuclease fragments of chloroplast DNA. The most abundant in vitro transcripts hybridize to chloroplast DNA fragments coding for 23 S, 16 S, and 5 S rRNAs. Only the coding strands of the rRNA genes are transcribed. Non-rDNA sequences of chloroplast DNA are also selectively transcribed but at much lower levels. The transcriptionally active chromosome has proved to be an ideal biochemical preparation for the study of selective transcription of cell organelle DNA.
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PMID:Selective in vitro transcription of Euglena chloroplast ribosomal RNA genes by a transcriptionally active chromosome. 676 27

Compounds of general structure N(5')pn(5')N were used by the reovirus-associated RNA polymerase as primers for template-directed synthesis of virus-specific oligonucleotides and RNA. Structural requirements for activity included a guanosine residue and at least four phosphates--i.e., G(5')pppp(5')N. Gp4G incubated with viral cores in the presence of CTP yielded Gp4GpC and CpGp4GpC. In a complete transcription mixture, Gp4G was also incorporated into the 5' termini of full-length transcripts in the unmethylated forms Gp4GpC and CpGp4GpC, in contrast to viral mRNAs that contain 5'-terminal m7GpppGmpC formed de novo. Gp5G, Gp6G, and Gp4A but not Gp2G, Gp3G, and Ap4A also primed reovirus transcription and inhibited RNA methylation.
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PMID:Priming of reovirus transcription by GppppG and formation of CpG(5')GpC. 695 5

The mechanism of streptolydigin inhibition of RNA synthesis has been investigated with a combination of steady state kinetics and product analysis by employing the abortive initiation reaction of Escherichia coli RNA polymerase. The pattern of inhibition by streptolydigin on the poly[d(A-T)] . poly[d(A-T)]template (non-competitive versus AMP; competitive versus UTP) was consistent with one inhibitor binding site and with an ordered addition of AMP followed by UTP. The more complicated patterns observed on the poly[d(I-C)] . poly[d(I-C)] template and the bacteriophage T7 A2 promotor (noncompetitive versus CTP) were explained by assuming that streptolydigin could stabilize the translocated ternary complex containing the product dinucleotide. Product analysis of two other abortive initiation reactions showed that the product did not dissociate from the inhibitor-bound translocated ternary complex. Finally, rifampicin and streptolydigin were shown to be functionally independent during initiation on several templates.
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PMID:On the mechanism of streptolydigin inhibition of Escherichia coli RNA polymerase. 698 76


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