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)

When cells infected with the Semliki Forest virus (SFV) mutant ts-4 were shifted to the nonpermissive temperature, synthesis of 26S RNA ceased, whereas synthesis of 42S RNA continued normally. These two single-stranded SFV RNAs are synthesized in two types of replicative intermediate (RI), 26S RNA in RI(b) and 42S RNA in RI(a). Cessation of 26S RNA synthesis after shift up in temperature was accompanied by loss of RI(b). When infected cells were shifted back down to 27 degrees C, 26S RNA synthesis resumed, coincident with the reappearance of RI(b). In both types of RI, the 42S minus-strand RNA is template for synthesis of plus-strand RNA. In pulse-chase experiments, we obtained RIs labeled only in their minus-strand RNA, and thus could follow the fate of RIs assembled at 27 degrees C when they were shifted to 39 degrees C. Our results show that, after shift up to 39 degrees C, there was a quantitative conversion of RIs in which 26S RNA had been synthesized to RIs in which 42S RNA was synthesized. This conversion of RI(b) to RI(a) was reversible, since RIs in which 26S RNA was synthesized reappeared when the infected cultures were shifted back down to 27 degrees C. We propose that, associated with RI(b), in which 26S RNA is synthesized, there is a virus-specific protein that functions to promote initiation of 26S RNA transcription at an internal site on the 42S minus-strand RNA and to block transcription on the minus strand in this region by the SFV RNA polymerase that had bound and was copying the minus-strand RNA from its 3' end. A ribonuclease-sensitive region would thus result in the sequence adjacent to the one that was complementary to 26S RNA. This virus-specific protein is not a component of the SFV RNA polymerase that continues to transcribe 42S RNA, and it is temperature sensitive in ts-4 mutant-infected cells. When this virus-specific protein is not present on RIs, the SFV polymerase transcribes the whole 42S minus-strand RNA and yields 42S plus-strand RNA.
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PMID:Mechanism for control of synthesis of Semliki Forest virus 26S and 42s RNA. 62 75

Adenovirus specific 5.5S RNA is heterogeneous at its 5' terminus. Complete pancreatic RNase digests of the RNA reveal a 5' terminal oligonucleotide (pp)pApGpCp in addition to the major 5' terminal (pp)pGpGpGpCp (1). Both 5' termini are detected early as well as late after adenovirus infection. In isolated nuclei, alpha-amanitin inhibits all 5.5S RNA transcription at a concentration of 200 microgram/ml, indicating that both initiation sites are recognized by RNA polymerase III.
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PMID:Two initiation sites for adenovirus 5.5S RNA. 64 8

DNA-dependent RNA polymerase class C (or III) has been solubilized from either uninfected or adenovirus-2-infected HeLa cells and purified by chromatography on phosphocellulose, DNA-cellulose, CM-Sephadex and DEAE-Sephadex. The last column separated the enzyme into three forms CI, CII and CIII, which were completely free of RNA polymerases class A and B and of DNase and RNase. The total and the relative amount of these different enzyme C forms did not vary whether purified from uninfected or infected cells. Irrespective of the stage of purification, the three enzyme forms transcribed deproteinized adenovirus-2DNA very efficiently. This transcription was highly sensitive to elevated ionic strength (especially in the presence of Mg2+) and was accompanied by continuous reinitiation as shown by adding poly(rI), a potent inhibitor of initiation. In addition heparin-resistant initiation complexes could be formed at elevated temperature. The RNA synthesized in vitro on deproteinized intact adenovirus-2 DNA by the different forms of RNA polymerase class C, has been characterized. Analysis of the transcripts by gel electrophoresis, RNA self-annealing, hybridization to separated adenovirus-2 DNA strands and to restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that the various regions of the adenovirus-2 genome were randomly transcribed. In addition, hybridization of RNA transcripts labelled at their 5' end by either [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected). The results are discussed in terms of the components which are possibly involved in specific transcription.
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PMID:Transcription in vitro of adenovirus-2 DNA by RNA polymerases class C purified from uninfected and adenovirus-infected HeLa cells. 71 Apr 51

RNA transcribed in vitro at low ionic strength, from either rat liver chromatin or DNA, contains a significant amount of structure resistant to RNase in high salt buffer. This is observed with rat liver (form B polymerase) as well as with Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate: RNA nucleotidyltransferase; EC 2.7.7.6). Treatment with RNases specific for either double-stranded or hybrid RNA indicates that resistance to RNase is due to the presence of double-stranded RNA sequences. Denaturation kinetics in the presence or absence of RNase suggest that these sequences are formed by intramolecular base pairing. Their mean length is about 20 to 30 nucleotides, but 15-20% are more than 100 nucleotides long. They contain 60-65% G-C base pairs. The proportion of double-stranded segments is higher in chromatin transcripts than in DNA-templated RNA, and is higher with homologous RNA polymerase than with the bacterial enzyme. On the other hand, chromatin endogenous RNA polymerase, which is unable to initiate transcription, does not synthesize double-stranded RNA. The problem of the location of these sequences is discussed; preliminary results suggest that the 5' end of the RNA transcripts could be enriched in complementary sequences.
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PMID:Double-stranded RNA in chromatin transcripts formed by exogenous RNA polymerase. 77 79

Methods are developed for studying RNA molecules bound directly to DNA in bacterial nucleoids. It is found that among the 1000-3000 nascent RNA chains that normally are attached to the DNA via their associated RNA polymerase molecules, 74 +/- 14 chains per nucleoid can be bound differently. These chains unlike the other nascent RNAs remained bound to the DNA after the chromosome was deproteinized and sheared. Sensitive assays using radioactive labels detected no RNA polymerase involved in the RNA-DNA linkage. The linkage was stable at low temperatures, but the RNA separated from the DNA at high temperature. The bound RNA molecules were heterodisperse (weight average length 1200 bases). Pulse-chase experiments and studies of the fate of these RNA molecules in rifampicin treated cells demonstrated that they are nascent RNAs, degraded or released from the DNA in vivo with kinetics similar to that of the total nascent RNA. Hybridization analyses showed that the chains are composed at least in part of nascent rRNA and known mRNA molecules. Some, but not more than 5% of the bound chains, contained sequences of about 300 nucleotides in length, bound to the DNA in an RNase resistant form.
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PMID:Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli. 77 42

Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.
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PMID:Hexamer of bacteriophage f2 coat protein as a repressor of bacteriophage RNA polymerase synthesis. 80 44

A RNase from calf thymus, which specifically cleaves native or synthetic double-stranded RNA molecules endonucleolytically, has been isolated and purified from calf thymus. For optimal activity, the enzyme requires a sulfhydryl reagent and divalent cations; over 95 per cent of the activity is inhibited by 0.5 mm ethidium bromide. The degradation of [3H]poly(C)-poly(I) by purified enzyme preparations yields labeled dinucleotides and octanucleotides; the latter oligonucleotide contained 5'-phosphate and 3'-hydroxyl termini. The enzyme cleaves high molecular weight RNAs such as RNA products formed in vitro by T3 phage-induced RNA polymerase from T3 phage DNA, heterogeneous RNA isolated from duck reticulocyte nuclei, and 45 S RNA isolated from rat liver nucleoli. The mode of degradation of RNA in vitro with the double-stranded RNase is similar to that of Escherichia coli RNase III and appears to act endonucleolytically. The degradation of 45 S RNA with the enzyme results in the production of 29 S and 19 S RNA fragments. These findings suggest that the enzyme may be involved in the processing of high molecular weight precursor RNAs to mRNA or rRNAs in a manner analogous to that reported for RNase III of E. coli.
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PMID:Isolation and purification of double-stranded ribonuclease from calf thymus. 83 40

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

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


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