Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:003721 (Poly)
 11,742 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several in vitro properties of partially purified form II RNA polymerase from Drosophila melanogaster embryo nuclei are described. The enzyme preparation is free from contaminating RNase, protein kinase, and polyphosphate kinase activities and can be used to study the incorporation of gamma-32P-labeled nucleoside triphosphates. The enzyme exhibits a biphasic heat inactivation pattern which is probably related to differential lability of its two subforms. However, a considerable protection against heat inactivation is provided by the nucleoside triphosphates present in the in vitro reaction system such that the enzyme catalyzes RNA synthesis in a nearly linear mode for over 2 hr at 30 C. Two initiation inhibitors, rifamycin AF/013 was found unsuitable for critical studies because of the high concentrations necessary for total inhibition (200 micrograms/ml) and particularly because of the obligate use of solvents which secondarily have a destabilizing effect on native DNA. Poly[I] was found to effectively block initiation at very low concentrations (1 microgram/ml). The enzyme rapidly forms poly[I]-resistant preinitiation complexes on both double- and single-stranded DNA. These complexes decay with a half-life of 2.5--3 min. RNA synthesis from poly[I]-resistant complexes amounts to 10% of the total potential synthesis on both double- and single-stranded DNA. Enzyme-DNA saturation experiments indicate that the form II enzyme discriminates two types of sites on Drosophila DNA, tight binding and weak binding, from which RNA synthesis proceeds slowly and rapidly, respectively. The tight-binding sites appear to be analogous to those sites with which the enzyme is able to form poly[I]-resistant complexes.
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PMID:Form II DNA-dependent RNA polymerase from Drosophila melanogaster: general in vitro catalytic properties and template interactions. 11 Mar 17

Poly(2'-fluoro-2'-deoxyuridylic acid) is known to be an effective inhibitor of the deoxyribonucleic acid polymerase found within the oncornaviruses. This synthetic polynucleotide was found to inhibit the replication of vesicular stomatitis virus in mouse L cells. The polymer was shown to be capable of inhibiting the viral ribonucleic acid (RNA)-dependent RNA polymerase, and it is proposed that this is the mechanism of antiviral activity. The following observations support this viewpoint: (i) the polymer is most active when added after virus adsorption; (ii) the antiviral activity is not species specific; and (iii) the polynucleotide is nontoxic to the host cell. Conventional methodologies designed to increase nucleic acid uptake by cultured cells do not show an increase in antiviral potency.
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PMID:Inhibition of vesicular stomatitis virus replication by poly(2'-fluoro-2'-deoxyuridylic acid). 17 88

Progesterone causes in goblet cells of oviducts of estrogen hormone-stimulated immature quails selectively gene activation without affecting DNA synthesis. This biological model has been used to study the influence of poly ADP-ribosylation during the processes of DNA transcription. Administration of progesterone in vivo causes an increase of the activity of RNA polymerase I and II in isolated nuclei. This increase is accompanied by a marked decrease of the specific activity of poly (ADP-Rib) polymerase. After in vitro ADP-ribosylation of nuclear proteins the template capacity of chromatin for ""exogenous'' RNA synthesis (with E. coli DNA-dependent RNA polymerases) as well as for ""endogenous'' RNA synthesis with DNA dependent RNA polymerases II is not affected, whereas the data presented seem to indicate that the capacity for RNA synthesis mediated by ""endogenous'' DNA-dependent RNA polymerase I might be inhibited after ADP-ribosylation. Evidence is presented to show that a considerable amount of poly (ADP-Rib), synthesized by poly (ADP-Rib) polymerase in isolated nuclei, is linked with RNA polymerase I. The rate of synthesis of poly (ADP-Rib) is dependent on the incubation temperature (optimum at 25 degrees C) and it can be inhibited by the specific inhibitors of poly (ADP-Rib) polymerase nicotineamide, thymidine and formycin B. Poly (ADP-Rib) is probably associated with RNA polymerase I through a covalent linkage. ADP-ribosylated RNA polymerase I has been purified 550 fold with respect to the nuclear extract corresponding to a 4,000 fold purification from the whole cell homogenate. The ratio between poly (ADP-Rib), formed during preincubation of nuclei with NAD, and RNA polymerase I remains almost constant during the purification procedures. The extent of ADP-ribosylation of RNA polymerase I decreases during gene expression. Thus we conclude that poly ADP-ribosylation of this enzyme is one of the regulatory mechanisms by which specificity of DNA transcription is achieved.
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PMID:Poly ADP-ribosylation of DNA-dependent RNA polymerase I from quail oviduct. Dependence on progesterone stimulation. 18 9

A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.
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PMID:Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). 19 96

The RNA-dependent RNA polymerase associated with vesicular stomatitis virus has been found to be markedly inhibited at high concentrations of virus. This endogenous inhibitor of the virion transcriptase was completely reversed by the action of two negatively charged polyamino acids: poly(L-glutamic acid) and pepsin (EC 3.4.23.1). Two other polyanions, heparin and polyethylene sulfonate, strongly inhibited the activity of the virion transcriptase even at low virus concentrations. Poly (L-glutamic acid) rapidly released the block in transcription of concentrated vesicular stomatitis virus, possibly owing to competition for binding sites of the inhibitor on the virion nucleocapsid transcription complex.
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PMID:Reversal by certain polyanions of an endogenous inhibitor of the vesicular stomatitis virus-associated transcriptase. 20 38

An endogenous transcriptase inhibitor active at high concentrations of vesicular stomatitis (VS) virus was present in trypsinized whole virions but was absent from ribonucleoprotein cores containing only the L, N, and NS proteins. Poly(L-glutamic acid) effectively reversed the transcriptase inhibition. Transcription under noninhibited, inhibited, and poly(L-glutamic acid)-reversed conditions did not appear to greatly affect the nature of the RNA transcription product. The VS virion matrix (M) protein was purified to greater than 98% homogeneity and was found to have an isoelectric point of approximately 9.0. Purified M protein inhibited transcription by ribonucleoprotein cores, an effect that was partially reversed by poly(L-glutamic acid). Two group III temperature-sensitive (ts) mutants of VS virus (tsO23 and ts G31) with lesions in the M protein exhibited little or no endogenous inhibitor activity compared with two wild-type strains and a group V mutant (tsO45) with a lesion in the G protein. The data presented strongly suggest that the virion M protein is responsible for the endogenous inhibition of in vitro RNA synthesis seen at high concentrations of VS virus.
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PMID:Role of the membrane (M) protein in endogenous inhibition of in vitro transcription by vesicular stomatitis virus. 21 13

Exposure of sulfhydryl groups as indicated by titration kinetics is decreased under conditions where RNA polymerase exists as a dimer or higher aggregate (low salt), in the presence of Mn2+, or when bound to d(A-T). Incubation of phenylmercurisulfonate with RNA polymerase above pH 9.0 results in loss of d(A-T) binding ability. Poly(U) binding is more sensitive to sulfhydryl modification and is lost as pH's above 8.0. The presence of 4 mM Mn2+ has an obvious effect in stabilizing the polymerase-poly(U) complex when incubated with 10 muM phenylmercurisulfonate + 1 M urea. Incubation of the enzyme with the mercurial and urea results in disaggregation to subprotomeric forms and release of the alpha subunit. Similar treatment in the presence of 4 mM MnSO4 stabilizes the protomeric structure of the enzyme. During chain elongation the enzyme exists as a ternary d(A-T)n-enzyme-r(U-A)n complex in which the bound d(A-T)n is refractory to the destabilizing effect of the mercurial; however, further phosphodiester bond formation is inhibited. The results are defined in terms of a role which reflects the involvement of polymerase sulfhydryl groups in the various conformations necessary for subunit-subunit interaction, tight template binding and catalytic activity.
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PMID:On the role of sulfhydryl groups in the structure and function of the Azotobacter vinelandii RNA polymerase. 24 Apr 5

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.
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PMID:Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter. 63 67

Poly(A)-poly(U) synthesis in the absence of template DNA is a unique reaction catalyzed by the RNA polymerase [EC 2.7:7.6] holoenzyme II of Escherichia coli. As one approach to investigating the physiological role of the enzyme, the molecular mechanism of poly(A)-poly(U) synthesis was studied. Streptolydigin, an inhibitor of the elongation of RNA chains, was shown to inhibit poly(A)-poly(U) synthesis, and the inhibition was released by a streptolydigin-resistant mutation on the beta subunit. These observations indicate that the active site for the reaction might be located on the beta subunit. Another antibiotic, rifampicin, which is known to be a specific inhibitor of the initiation of DNA-dependent RNA synthesis, effectively inhibited both initiation and elongation steps in poly(A)-poly(U) synthesis. This suggests that the enzyme conformation during the chain elongation reaction might be different in this case from that in DNA-dependent RNA synthesis. Analysis of the products formed during the initiation reaction indicated that the rate-determining reaction in poly(A)-poly(U) synthesis was the formation of primers of short chain length, and that holoenzyme I was unable to form the first phosphodiester bond in this reaction. Functional properties of holoenzyme II are discussed in connection with these observations.
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PMID:Mechanism of polyadenylate-polyuridylate synthesis by RNA polymerase holoenzyme II of Escherichia coli. 78 Mar 49

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


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