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)

We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.
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PMID:Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA. 302 98

In order to determine the mechanism and enzyme(s) responsible for 3' processing of tRNA precursors, we have developed an in vitro processing system that uses as substrates two SP6 RNA polymerase-generated transcripts of the gene for tRNA(Tyrsu3)+ that contain 49 extra 5'-nucleotides and either 5 or 25 extra 3'-nucleotides. A high speed supernatant fraction from an Escherichia coli strain deficient in five ribonucleases was found to accurately process both tRNA precursors in this system to the size of mature tRNA(Tyr). Final 3' end processing of each precursor occurs in an exonucleolytic manner to generate the correct 3' terminus; however, a prior endonucleolytic cleavage also is observed in processing of the longer precursor. The system requires Mg2+ and works optimally at about 50 mM KCl and pH 8-9. Dialysis of the supernatant fraction leads to loss of processing activity but can be restored to normal by the addition of inorganic phosphate or arsenate. Furthermore, nucleoside diphosphates are a product of the processing reaction. These data indicate that 3' processing in RNase-deficient extracts involves a phosphorolytic reaction. On the other hand, phosphate is not required for processing in RNase+ extracts, although it does aid in processing of the longer precursor. The usefulness of this in vitro system for studies of tRNA processing and the identity of the phosphate-requiring enzyme are discussed.
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PMID:3' processing of tRNA precursors in ribonuclease-deficient Escherichia coli. Development and characterization of an in vitro processing system and evidence for a phosphate requirement. 327 67

A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined.
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PMID:Isolation and characterization of DNA-dependent RNA polymerase III from Leishmania mexicana and inhibition by purine analogs. 343 22

The binding sites for influenza viral RNA polymerase on genome RNA segments were investigated. Ribonucleoprotein (RNP) cores containing the RNA polymerase were isolated from detergent-treated virions by glycerol gradient centrifugation. On ApG-primed in vitro transcription by the isolated RNP cores, different levels of RNA transcripts were synthesized for the eight RNP cores, suggesting an uneven distribution of the RNA polymerase. 3'-Terminal labeling of the RNP cores with the use of [32P]pCp and T4-RNA ligase indicated a reciprocal correlation between the levels of the RNA-3' label and RNA synthesis. Centrifugation of detergent-treated virions in a double gradient of cesium trifluoroacetate (or cesium chloride) and glycerol yielded RNA polymerase-RNA complexes devoid of NP, the major RNA-bound protein, but the pattern of RNA-3' labeling remained virtually unaffected. All these observations together indicated that the RNA polymerase is associated near the 3' termini of some viral RNA segments, thereby preventing the in vitro labeling of the RNA-3' ends. The results of foot-printing experiments using RNase V1 and RNase T2 were in agreement with this model.
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PMID:Identification of the RNA polymerase-binding site on genome RNA of influenza virus. 343 66

Phage 82 gene Q encodes a phage-specific positive regulator of late gene expression, thought, by analogy to the corresponding gene of phage lambda, to be a transcription antiterminator. We have cloned and sequenced the phage 82 gene Q and have overproduced and purified the 82 Q protein. We also have identified and sequenced DNA containing the phage 82 late gene promoter and terminator. We show that purified 82 Q protein is active and specific for DNA containing the 82 late gene promoter in a well defined in vitro transcription reaction: RNA polymerase initiating at the phage 82 late gene promoter and modified by 82 Q protein reads through a downstream transcriptional terminator. We used T1 RNase mapping to confirm that the putative readthrough RNA made in the presence of 82 Q protein is in fact an elongation product of the shorter RNA.
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PMID:Bacteriophage 82 gene Q and Q protein. Sequence, overproduction, and activity as a transcription antiterminator in vitro. 362 33

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.
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PMID:Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract. 367 Mar 7

The foot-and-mouth disease virus-RNA polymerase complex was released from membrane particulates present in the cytoplasm of infected baby hamster kidney cells. The soluble polymerase complex was fractionated by zonal centrifugation in sucrose gradients. Two polymerase complexes (RNA and protein complex) active in the cell-free system were isolated and had S-rate ranges of 20-70S and 100-300S, respectively. The light polymerase complex contained 20S double-stranded RNA; and the heavy polymerase complex contained a polydisperse, partially RNase-resistant RNA. The cell-free product of these two polymerase complexes was analyzed by zonal centrifugation in sucrose gradients. The light polymerase complex synthesized only 20S double-stranded RNA. The product of the heavy polymerase complex contained no detectable 20S double-stranded RNA and only a peak of single-stranded RNA with S-rate corresponding to 37S viral RNA. A third polymerase complex was isolated with S-rate greater than 300S, and it contained a polydisperse, partially RNase-resistant RNA. This third polymerase complex synthesized both 37S viral RNA and 20S double-stranded RNA in the cell-free system, and it is probably the native polymerase complex still bound to cellular particulates.
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PMID:The isolation of two enzyme-ribonucleic acid complexes involved in the synthesis of foot-and-mouth disease virus ribonucleic acid. 430 96

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg(2+) or Mn(2+)) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T(1), inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T(1) RNase.
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PMID:Poly(A) polymerase from vaccinia virus-infected cells. I. Partial purification and characterization. 441 6

3T6 mouse fibroblasts were grown in 5-bromodeoxyuridine (BrdU) so that approximately 20% of the thymine residues in DNA were replaced by BrdU. BrdU replacement caused an alteration in the relative incorporation of labeled nucleotide precursors into RNA. The RNA synthesized by cells grown in BrdU has a lower proportion of adenine and a higher guanine complement. This was shown for (a) nascent RNA made in vivo by confluent monolayers of cells in culture; (b) RNA synthesized in vitro on a chromatin template with either homologous or heterologous RNA polymerase; and (c) RNA synthesized in vitro on a DNA template with a highly purified RNA polymerase. The product was completely digested by RNase. The relative decrease in the incorporation of adenine into RNA was reserved when BrdU-treated cells were allowed to proliferate in BrdU-free medium.
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PMID:Effect of 5-bromodeoxyuridine on chromatin transcription in confluent fibroblasts. 459 91

An enzymatic activity which synthesized oligo(A) in vitro was found in highly purified reovirus. The poly(A) polymerase activity was dependent on Mn(2+) and utilized only ATP, whereas the virion-associated RNA polymerase required all four ribonucleoside triphosphates and Mg(2+). Oligo(A) synthesis was demonstrated with complete virions and infectious subviral particles derived from virus by limited chymotrypsin digestion but not with cores, a product of extensive chymotrypsin digestion of virus. The enzymatic product and the oligo(A) from purified virions were isolated by binding to oligo(dT)-cellulose columns. Most of the in vitro product was similar in size and structure to the oligo(A) from purified virions by the criteria of gel electrophoresis, DEAE-cellulose chromatography, end-group analysis, and sensitivity to RNase. The evidence suggests that oligo(A) synthesis is mediated by the poly(A) polymerase during a late step in viral morphogenesis and may result from an alternative activity of the virion-associated transcriptase.
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PMID:Poly(A) polymerase activity in reovirus. 483 12


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