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)

High-molecular-weight native mouse DNA was transcribed with Escherichia coli RNA polymerase under low salt conditions, and the nature of the DNA sequences transcribed determined by molecular hybridization. The results indicated that E. coli RNA polymerase does not transcribe the sequences in native mouse DNA randomly under these conditions. First, hybridization with a large excess of mouse DNA showed that no more than 5% of the RNA synthesized had been transcribed from repeated sequences in the DNA. Second, hybridization with tracer amounts of labelled non-repeated mouse DNA indicated that the bulk of the RNA had been transcribed from less than 1% of the non-repeated sequences and only about 10% had been transcribed from a further 25% of these sequences; the remaining non-repeated sequences in the DNA, amounting to 50% of the genome, were not represented in the RNA synthesized in vitro to any detectable extent. Third, the proportion (40%) of complementary DNA transcribed from mouse-liver nuclear polyadenylated RNA which hybridized with the RNA synthesized in vitro was significantly greater than would have been expected if transcription had been random. The data have also been interpreted as indicating the presence of two types of initiation site for E. coli RNA polymerase in the non-repeated sequences in mouse DNA. The frequencies of their occurrence have been calculated to be one per 150 000 base-pairs and one per 500 base-pairs, respectively.
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PMID:Characterization of the RNA transcribed in vitro from native mammalian DNA by Escherichia coli RNA polymerase. 79 29

Nucleic acid-free extracts of Escherichia coli have been analyzed by chromatography on columns of cellulose, to which poly(A), poly(U), or poly(C) have been attached by ultraviolet irradiation. Proteins are released from the columns by stepwise elution with increasingly higher concentrations of salt, followed by washing with urea to remove very tightly bound molecules. The pattern of protein elution is reproducibly different for each of the homopolymer RNA-cellulose columns used: some proteins bind very tightly to one column, but poorly to others. Analysis by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, by immunological cross-reactivity in double diffusion tests, and by enzymological assays, has allowed the identification of a number of these proteins. The RNA polymerase core enzyme binds to poly(C)- and to poly(U)-cellulose columns, and can be purified to 20 to 30 percent homogeneity in a single step. Ribosomal protein S1 and the termination factor rho bind very tightly to poly(C)-cellulose, and both can be purified to homogeneity rapidly, in much higher yields than previously reported. Poly(A)-cellulose chromatography allows the isolation of large amounts of an 80,000 molecular weight protein having an as yet unassigned cellular function. The host factor required for RNA phage Qbeta RNA replication in vitro can also be obtained from poly(A)-cellulose, and chromatography of extracts of phage Qbeta-infected E. coli on RNA-cellulose columns results in very rapid isolation of the Qbeta replicase enzyme. Homopolymer RNA-cellulose chromatography thus appears to be a simple, general technique, useful for the efficient isolation of a variety of RNA-binding proteins.
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PMID:Isolation of bacterial and phage proteins by homopolymer RNA-cellulose chromatography. 80 80

The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration. RNA was transcribed (at 0.25 M KCl concentration) from high-molecular-weight mtDNA which was then translated in an E. coli (S-30) cell-free protein synthesising system. Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems. Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes. Electrophoresis of the completely dissociated antigen-antibody complexes using dodecylsulphate-polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide-inhibited whole cells.
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PMID:Synthesis of mitochondrial proteins in an Escherichia coli cell-free system directed by yeast mitochondrial DNA. 80 75

Two species of form A (or I) RNA polymerase have been identified in eucaryotic cells and there is evidence that this alpha-amanitin-insensitive activity exists as two discrete pools: a pool of 'free' activity, which is identified by its ability to transcribe poly d(A-T) in the presence of actinomycin in vitro, and a pool of enzyme in the form of a transcription complex ('engaged') which is unaffected by inhibitors of initiation of RNA synthesis. 1. The principles underlying and the practical application of the technique used to define the pool of 'free' RNA polymerase activity have been analysed in considerable detail. On the basis of actinomycin titrations of poly[d(A-T)]dependent activity in isolated organelles, it is concluded that a pool of 'free' RNA polymerase A activity exists in mammalian nuclei which, under certain circumstances, is lost from nuclei during their isolation. The evidence presented suggests that nucleoli, resolved from nuclei by the classical sonication technique, contain form A polymerase exclusively in the transcription complex form. 2. Different techniques used to solubilise RNA polymerase activity from nucleoli are shown to give rise to different proportions of the two form A RNA polymerase species (AI and AII, as defined by their differential elution from phosphocellulose): whereas low-ionic-strength extraction gives rise to form AII, high-salt, sonication extracts contain predominantly the form AI enzyme. It is shown that the sonication technique results in the conversion of form AII to form AI. By a careful appraisal of the products of these procedures and a novel polymerase solubilisation technique, it is concluded that RNA polymerase AII is the 'engaged' form of the enzyme found in the transcription complex. 3. Making use of the finding that the 'free' form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity has been characterised without the requirement for solubilisation techniques which might result in the conversion of one form to another: the 'free' species is shown to be form AI RNA polymerase. 4. These conclusions that two discrete pools of form A RNA polymerase activity contain different species of the enzyme are briefly discussed in the light of other published information concerning their subunit structures and their potential role in the expression of the ribosomal RNA coding sequences.
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PMID:Forms AI and AII DNA-dependent RNA polymerases as components of two defined pools of polymerase activity in mammalian cells. 83 29

Although the bulk of RNA synthesized in vitro by vaccinia virus is 8 to 12 S, a small amount of high molecular weight RNA can be detected. This RNA is virion-associated and is not extruded from the virus as high molecular weight RNA. It is sensitive to pancreatic RNase digestion in high salt, has a density in neutral CS2SO4 of 1.68 g ml-1 and remains large after digestion with DNase or denaturation in dimethyl sulfoxide. In the presence of high concentrations of virus in the in vitro RNA polymerase reaction, pulse-labeling experiments indicate an RNA sedimenting heterogeneously between 20 and 30 S. Pulse-chase experiments indicate that a fraction of this high molecular weight RNA can be chased into RNA sedimenting at 8 to 12 S. Cleavage into smaller fragments is not dependent on continued RNA synthesis but does require ribonucleoside triphosphates. In the presence of ethidium bromide, the RNA is not cleaved.
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PMID:In vitro synthesis of a high molecular weight virion-associated RNA by vaccinia. 83 1

alpha-Amanitin-resistant clones were selected in the mouse lymphoblastoid cell line L5178Y. One resistant clone, named A169b, was recloned and the properties of its DNA-dependent RNA polymerases were examined. The RNA polymerase II activity from A169b differs from the parental cell line in that approximately half the activity is resistant to 0.5 microgram/mL alpha-amanitin, while the parental enzyme is 50% inhibited at 0.005 microgram/mL. The enzymes from A169b and the parental line were purified free of polymerase III and their properties compared. The two preparations were identical in their apparent affinities for the four nucleoside triphosphates, in their salt and divalent cation preferences, and in their preference for denatured over native DNA. They differed in their response to alpha-amanitin. The apparent K1 for the parental enzyme was 3.5 X 10(-9) M; plots of 1/V vs. alpha-amanitin concentration gave a biphasic curve with A169b enzyme. The two apparent K1 values were 4.1 X 10(-9) and 2.1 X 10(-6) M. In addition, the enzyme from A169b showed a twofold higher activity on poly [d(AT)] as template, compared to native DNA, than that of the parental enzyme. Other template preferences may be affected, but differences were marginal. These results indicate that mutation to alpha-amanitin resistance may alter other enzymatic parameters; such mutations may be helpful in elucidating structure-function relationships in these complex enzymes.
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PMID:Properties of an altered RNA polymerase II activity from an alpha-amanitin-resistant mouse cell line. 90 71

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.
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PMID:Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin. 94 34

DNA-dependent RNA polymerase C, partially purified from Xenopus laevis ovaries, has been resolved by DEAE-Sephadex chromatography in two forms, eluting at 0.2 M and 0.3 M ammonium sulfate, respectively. Both are sensitive to high concentrations of alpha-amanitin (200 mug/ml). Their ionic strength dependence and divalent cation requirements are indistinguishable. Quantitatively, RNA polymerase C represents the major form of RNA polymerase activity solubilized from the ovaries. Both RNA polymerases C are able to transcribe efficiently either high-molecular-weight Xenopus DNA or intact adenovirus DNA, as compared to nicked DNA. In contrast, RNA polymerase A has little activity on an intact DNA template. The salt dependence of the RNA polymerases C activity is different on the two kinds of template. Nicked DNA is efficiently transcribed up to a salt concentration of 100 mM ammonium sulfate. On intact DNA, optimal transcription is obtained at 40 mM ammonium sulfate and is inhibited by higher salt concentrations.
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PMID:DNA-dependent RNA polymerase C from Xenopus laevis ovaries. Ability to transcribe intact double-stranded DNA. 94 51

Factors influencing promoter site selection by Escherichia coli RNA polymerase were investigated using T7 DNA as template. The utilization of the three major early promoters A1, A2 and A3, was followed by processing the RNA transcripts with RNAase III, which generates the three corresponding initiator RNA fragments. The three promoters proved to be functionally distinct. A strong differential effect of temperature and ionic strength on promoter selection was observed. The transition temperature of promoters A1, A2 and A3 was measured directly by preincubating, at different temperatures, RNA polymerase and T7 DNA, with primer-substrate combinations which selected each site independently. The transition temperature of the three sites was markedly different. Promoter A3 was used predominantly at low temperature, whereas A1 or A2 promoters were gradually activated by increasing the temperature. The temperature response curves were strongly dependent upon the salt concentration. On the other hand, challenge experiments with rifampicin or poly (inosinic acid) showed that, once preinitiation complexes are formed by incubating RNA polymerase with DNA at 37 degrees C, the three sites A1, A2 and A3 promote chain initiation with equal efficiency and at the same rate.
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PMID:Interaction of RNA polymerase from Escherichia coli with DNA. Effect of temperature and ionic strength on selection of T7 DNA early promoters. 94 73

HeLa cell nuclei, isolated 17 h after infection with human adenovirus type 2 (Ad2), were treated with 200 mM ammonium sulfate. The extract (S200 fraction) contained 50 to 70% of the nonintegrated Ad2 DNA, which was in the form of nucleoprotein complexes. These complexes contained native, intact Ad2 DNA (with the exception of replicative intermediates) and could be partially purified and resolved by velocity gradient centrifugation. Using high-salt (200 mM ammonium sulfate) incubation conditions, more than 95% of the nuclear RNA polymerase activity belonged to class B. About 45% of the class B enzyme molecules bound to DNA in the nuclei (those "engaged" in RNA synthesis) were released from the nuclei in the form of Ad2 transcriptional complexes by treatment with 200 mM ammonium sulfate. At least 90% of the RNA synthesized in high salt in the nuclei or in the S200 fraction was Ad2 specific, and essentially all of this RNA was complementary to the l strand of Ad2 DNA. These findings are compatible with what is known about Ad2-specific RNA synthesis in vivo. The analysis of the RNA synthesized from partially purified transcriptional complexes supports the contention that its transcription is almost entirely asymmetric, and that the asymmetry observed in vivo is not a consequence of the rapid degradation of h-strand transcripts. The RNA synthesized in vitro in the absence of detectable RNase activity sedimented with a maximum size of 35 to 40S. Less than 5% of the nuclear or the S200 fraction RNA polymerase activity was class C when assayed under non-reinitiating conditions. Although much of the RNA synthesized by the class C enzyme was Ad2 specific, 5.5S virus-associated RNA was not the predominant product. The isolation of Ad2 DNA transcriptional complexes provides an attractive system for further characterizing the Ad2 DNA template used for transcription and for studying the regulation of the expression of the Ad2 genome during the productive infection cycle.
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PMID:Characterization of adenovirus type 2 transcriptional complexes isolated from infected HeLa cell nuclei. 95 Jun 90


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