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)

Nuclei from seminal vesicle epithelium of adult guinea pigs were isolated in hypertonic sucrose solution. The incorporation of [3H]UTP by the isolated nuclei into acid-precipitable products was studied. Incorporation required ATP, GTP, CTP, UTP, and Mg+2. It was inhibited by addition of actinomycin D, deoxyribonuclease, or pyrophosphate to the reaction mixture. Thus, incorporation of [3H]UTP by isolated nuclei had the same characteristics that have been demonstrated for the reactions catalyzed by nuclear RNA polymerases. Using alpha-amanitin as a metabolic tool, we established concentrations of (NH4)2SO4. Mg+2, and nucleotides that give maximum assayable activities of nuclear RNA polymerases I and II. When the activities of polymerases I and II were measured in isolated seminal vesicle nuclei of guinea pigs that had been castrated 4 days earlier, a marked decrease in activities was found relative to control values (nuclei from intact animals). No further decrease was found 8 days after castration. Diminished accessibility to the nuclear DNA template and a decrease in the concentration of RNA polymerase molecules seemed to be responsible for the observed effects of castration on activities of RNA polymerases. An increase in ribonuclease activity did not seem to be responsible for the effects of castration. Activities of the enzymes did not change 2, 3, or 4 hours after intraperitoneal injection (2 mg/kg body weight) of each of five different androgens. Similarly, a single intraperitoneal injection of testosterone did not restore enzyme activity of polymerade I or II at any time during the first 24-hour period after hormone administration.
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PMID:RNA polymerase activities in isolated nuclei of guinea pig seminal vesicle epithelium: influence of castration and androgen administration. 90 9

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

Nuclei of GH3 cells, isolated by detergent lysis, synthesized RNA for an extended period at 29 degrees C in the presence of rat liver ribonuclease inhibitor (RI). Extended RNA synthesis was dependent upon the presence of RI. Sucrose gradient sedimentation analysis of the cell-free reaction products showed that RNAs ranging from 4 S to greater than 28 S were synthesized. Further characterization of the RNA products was made by examining the sensitivity of synthesis to alpha-amanitin and actinomycin D as well as by oligo(dT)-cellulose binding properties. Evidence was obtained that RNA polymerases I, II, and III were functioning in isolated GH3 nuclei. Newly synthesized RNAs were found in both the nuclear pellet and postnuclear supernatant fractions. RNA polymerase I products remained associated with the nuclear pellet throughout a 60-min incubation period whereas RNAs synthesized by RNA polymerase III emerged rapidly into the supernatant fraction. RNA polymerase II products were distributed in both fractions and were found to contain poly(A). De novo poly(A) synthesis was demonstrated and found to be inhibited by cordvcepin triphosphate (3'-dATP). Supernatant RNAs synthesized by polymerase II contained a poly(A) segment of about 150 adenine residues; these transcripts sedimented heterogeneously with an apparent size distribution (under denaturing conditions) which was smaller than that of nuclear RNA polymerase II products and which resembled that of cellular mRNA. Qualitative differences in the nuclear and supernatant RNAs, the kinetics of appearance of the latter, and the differential effect of 3'-dATP on the extranuclear appearance of supernatant RNAs suggest that a process resembling nuclear-cytoplasmic RNA transport occurred in this cell-free nuclear system.
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PMID:Extended RNA synthesis in isolated nuclei from rat pituitary tumor cells. 98 56

Temperature-sensitive "leaky" mutants of phage MS2 having white dense ring around negative colonies are described. As these mutants are used for quantitative genetic studies, the white ring presents interest. Typical mutant 40 is used as a model for investigation. Light microscopy has shown, that cells from white ring zone have spore-like inclusions, which determine the characteristic structure of surrounding mutant negative colonies. Cytochemical reactions for the presence of glicogen, lipids, volutin, nuclear material and spores were negative. Electrone microscopy of negatively stained samples and ultrathin sections has revealed that cells from white ring zone, unlike phage-infected wild type cells, have two types of electron dense inclusions: 1) crystalline structures formed with great number of closely packed mature phage particles, and 2) large amorphic bodies. Electrone microscope-cytochemical data showed that inclusions remain intact under treatment of ultrathin sections of white zone ring with DNase and perchloric acid, while nuclear material was completely destroyed. Amorphic bodied were completely destructed after the treatment with RNase, while nuclear material and crystalline phage aggregated remained unchanged. Therefore, amorphic bodies consist of RNA, which has not been used to form virions. Single cycle of the development of mutant 40 at 37 degrees and 43 degrees C and under the temperature of incubation 37 degrees leads to 43 degrees C and 43 degrees leads to 37 degrees C in the course of intracellular reproduction is investigated. Influence of the phage on growth on infected culture is studied. The data obtained draw to a conclusion that the impaired function belongs to cystron protein of the phage membrane. As certain mutations in this cystrone of RNA-containing phage result in the depression of cystrone RNA polymerase, it is supposed that the formation of RNA containing bodies in infected cells, determining the formation of white rings in NA, together with cristalline aggregates of cells, is a result of mutation damage of cystrone protein of the phage MS2 membrane.
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PMID:[Effect of mutagens on RNA-containing phages and its infectious RNA. VII. Genetic nature of morphologic mutants of RNA-containing phage MS2]. 99 65

We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.
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PMID:In vitro synthesis of transfer RNA. I. Purification of required components. 109 89

We have shown that the synthesis of active su+III tRNATyr from a phi80psu+III DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-dependent RNA polymerase, catalyzes the formation of a large molecular weight transcript, initiating synthesis at a specific site 41 nucleotides proximal to the 5' end of the su+III tRNATyr structural gene and continuing at least 100 nucleotides beyond the 3' terminus of the su+III tRNATyr sequence. The second required component, designated Fraction V, allows purified DNA-DEPENDENT RNA polymerase to function in tRNA synthesis. We have shown that this fraction contains an endonuclease that together with DNA-dependent RNA polymerase is responsible for the synthesis of su+III tRNATyr "precursor". Thus, su+III tRNATyr precursor is not itself the primary transcription product of the su+III tRNATyr gene, but rather, it arises as a result of post-transcriptional cleavage of a much larger transcript by the action of the nuclease present in Fraction V. The third enzymatic activity required for synthesis of active su+III tRNATyr is a ribonuclease (RNase P III) that specifically catalyzes the removal of the 3' extra nucleotides from the su+III tRNATyr precursor. The fourth activity required for synthesis of tRNA is a previously identified endonuclease, RNase P, that specifically catalyzes the removal of the 5' extra nucleotides from tRNA precursors. The properties of RNase P purified according to the procedure developed in this laboratory have been compared with those of the enzyme purified from ribosomes according to the procedure described by Robertson et al. (Robertson, H.D., Altman, S., and Smith, F.D. (1972) J.Biol. Chem. 247, 5243-5251.).
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PMID:In vitro synthesis of transfer RNA. II. Identification of required enzymatic activities. 109 90

During chain elongation RNA polymerase exists as a ternary DNA-enzyme-RNA complex in which a discrete length of the nascent RNA chain proximal to the 3'-OH terminus will be bound to the product binding site (Krakow, J. S., and Fronk, E. (1969) J. Biol. Chem. 244, 5988). We have utilized the poly[d(A-T)]-directed reaction to determine the length of the nascent poly[r(A-U)] protected from attack by pancreatic ribonuclease. Following release of the ribonuclease resistant oligo[r(A-U)] from the ternary complex, its size was determined by ion exchange chromatography on DEAE-cellulose, gel filtration on Bio-Gel P-10, and the ratio of 3'-terminal uridine to internal 2':3'-UMP following alkaline hydrolysis. The results indicate that the length of the nascent protected fragment is approximately 12 residues.
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PMID:Studies on the product binding sites of the Azotobacter vinelandii ribonucleic acid polymerase. 112 30

DNA-dependent RNA polymerase was isolated from rat spleen cell nuclei and was identified as A and B RNA polymerases by data on DEAE- and P-cellulose ionic exchange chromatography and on concentration dependency on bivalent ions and (NH4)2SO4. Two forms of the enzyme differed from each other in the activity in RNA synthesizing system, and their activity was completely inhibited by actinomycin, DNase and RNase.
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PMID:[DNA-dependent RNA polymerase from the nuclei of the spleen of white rats]. 113 3

A single ip injection of triiodothyronine (T3; 30 mug/100 g BW) to thyroidectomized rats markedly stimulates RNA synthesis in isolated liver nuclei. The increased level of RNA synthesized in vitro by isolated nuclei does not depend on a reduced degradation of the nascent RNA molecules, since ribonuclease activities are not affected by the administration of T3. In addition, our results have confirmed previous findings of Tata et al. that the increase in nucleolar alpha-amanitin-resistant RNA polymerase I activity at low ionic strength always preceded the rise of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase II activity at high ionic strength. Moreover, it has been found that a significant increase in an alpha-amanitin-resistant activity at high ionic strength occurs as early as 10 h after hormone injection. This enzyme, which forms RNA with a U to G ratio significantly higher than that of RNA synthesized by the nucleolar alpha-amanitin-resistant enzyme, is probably nucleoplasmic RNA polymerase III which is though to synthesize 5S and transfer RNAs. The possible role and the mechanism(s) of the early and concomitant increase in nucleolar and nucleoplasmic alpha-aminitin-resistant activities, and of the subsequent rise of RNA polymerase II activity following T3 administration are discussed.
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PMID:Sequential stimulation of nuclear RNA polymerase activities in livers from thyroidectomized rats treated with triiodothyronine. 119 15

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.
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PMID:Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response. 123 May 9


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