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)

Bacteriphage T7 RNAs have been fractionated on preparative polyacrylamide gels. The in vitro coding capacities of the RNAs have been determined by translation of the RNAs in a cell-free system and analysis of the polypeptide products on sodium dodecyl sulfate polyacrylamide slab gels. The T7 early RNAs are fractionated according to their molecular weight and without intermolecular aggregation. Fractionation of the late T7 RNAs gives rise to 10 major RNAs, ranging in size from 0.29 X 10(6) daltons to 2.05 X 10(6) daltons. Five of these RNAs are polycistronic and overlapping species are present for some T7 proteins. In particular, the gene 10 protein, the major capsid protein, is translated from at least three mRNAs. The smallest of these gene 10 mRNAs is monocistronic. A second gene 10 mRNA also codes for the gene 9 protein, and a third gene 10 mRNA codes for both gene 8 and gene 9 proteins. The T7 gene 3.5 protein, a T7 lytic enzyme, is also translated from several differently sized mRNAs. Comparison with published data on in vitro transcription by T7 RNA polymerase suggestes that transcription from multiple initiation sites and cleavage of larger precursors are both involved in generating the late T7 transcripts we observe. The overlapping mode of transcription could serve to amplify certain gene products.
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PMID:Detection of polycistronic and overlapping bacteriophage T7 late transcripts by in vitro translation. 106 Nov 35

In order to obtain RNA polymerase preparations carrying the necessary specificity determinants to transcribe the delayed-early genes of bacteriophage T4, crude extracts of uninfected and T4-infected Escherichia coli were fractionated in glycerol gradients of low ionic strength. In contrast to the reported sedimentation behavior of the purified enzyme, the RNA polymerase activity in crude extracts of normal and infected cells sedimented heterogeneously over a wide range of sedimentation coefficients. When the "heavy" (24-33 S) and "light" (14-20 S) regions of the gradient were precipitated with ammonium sulfate and recentrifuged, the former split into two subfractions, one again sedimenting heavy and the other sedimenting light. The latter did not split under the same conditions. The resulting subfractions from uninfected cell extracts had different thermal thermal stabilities at 50 degrees (half-lives ranging from 2-3 to 25 min) while those from T4-infected cell extracts were very thermolabile (half-life of 1-2 min). All the subfractions were more active on T4 DNA than on calf-thymus DNA. They also formed rifampicin-resistant, RNA chain initiation complexes with T4 DNA. Based on the kinetics of heat inactivation with T4 and calf thymus DNAs as templates and preferential transcription of T4 DNA, it is proposed that the T4-infected cell enzymes prepared as described here harbor heat-labile initiation factor(s). During infection the heavy sedimenting RNA polymerase activity disappears after 2.5 min at 37 degrees. This appears to require phage-specific protein synthesis because (a) it does not happen in the presence of chloramphenicol and (b) it does not happen in T4 ghost-infected cells.
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PMID:Transcription of bacteriophage T4 genome in vitro. Heterogeneity of RNA polymerase in crude extracts of normal and T4-infected Escherichia coli B. 109 Dec 88

An improved method is described for the purification of the DNA-dependent RNA polymerase [ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6] from Escherichia coli. The method involves lysozyme-sodium deoxycholate lysis, low-speed centrifugation, precipitation with Polymin P, elution from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DNA-cellulose and Bio-Gel A 5m. RNA polymerase is purified to electrophoretic homogeneity in 2 days with a recovery of 45%, resulting in a yield of 250 mg of holoenzyme from 500 g of cells.
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PMID:A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. 110 52

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

Three forms of DNA-dependent RNA polymerase have been separated by chromatography of extracts of yeast-like cells and mycelium of the dimorphic fungus Mucor rouxii. Each of the three eznymes has been purified by means of protamine sulfate precipitation, ion exchange chromatography, affinity chromatography, and velocity sedimentation. Electrophoresis under denaturing conditions showed differences in the subunit compositions of all three purified enzymes. The properties of the enzymes from M. rouxii were similar to those of polymerases from other eukaryotic organisms. Denatured DNA was a better template than native DNA for all three enzymes but each enzyme had a distinct pattern of activities with different templates. Enzymes I and III displayed optimal activity with Mn-2gs the divalent cation and were stimulated significantly by Kcl and (NH4)2S04. Enzyme II had a greater activity with Mg-2gnd was only slightly stimulated by KCl and (NH4)2SO4. None of the enzymes were inhibited by cycloheximide or by rifampicin: all were inhibited by actinomycin C and rifampin AF/018: only enzyme II was inhibited by alpha-amanitin. No differences could be found in the properties of the same enzymes isolated from yeast-like cells or mycelium.
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PMID:Deoxyribonucleic acid-dependent ribonucleic acid polymerases in the dimorphic fungus Mucor rouxii. 111 77

The DNA-dependent RNA polymerase from Pseudomonas BAL-31, the host for bacteriophage PM2, has been purified 154-fold using differential centrifugation, chromatography on DEAE-cellulose, ammonium sulfate precipitation, and sucrose gradient centrifugations at low and high ionic strength. The resulting enzyme is free of enzyme activities which could interfere with transcription studies and is greater than 85% pure as judged by polyacrylamide gel electrophoresis. Like other bacterial RNA polymerases, its subunit structure is beta'beta sigma alpha2. From gel electrophoresis the beta', beta, and alpha subunits have approximately the same molecular weights as those from Escherichia coli, whereas the sigma subunit is 5% larger (89,000 vs. 85,000). A summation of the subunits yields a molecular weight of 485,000 for the holoenzyme. Like other bacterial RNA polymerases, it sediments as a monomer (15 S) at low ionic strength (0.065) and as a dimer (22 S) at high ionic strength (0.75). Its activity is stimulated three-fold by monovalent cations (K+,NH4+, NA+) with additional stimulation provided by divalent cations (Mg2plus, Mn2plus). The transcription of phage PM2 form I (supercoiled) DNA has an ionic strength optimum of 0.26 for continuous long-term synthesis, and over an ionic strength range of 0.09-0.46 "plateau-type" kinetics are not observed. The sigma subunit is required for optimal PM2 transcription. The enzyme is sensitive to the same inhibitors of transcription as the RNA polymerase from E. coli, it has a temperature optimum of 28 degrees, and it is 50% inactivated by heating 10 min at 41 degrees. It has template preference similar to E. coli polymerase and shows little preference for homologous templates. With various DNAs the order of template activities is T7 greater than PM2 I congruent to T4 greater than PM2 II (relaxed circular form) greater than lambda-c greater than calf thymus greater than BAL-31 DNA. Phage PM2 form I DNA is transcribed at a twofold greater rate than PM2 form II DNA by this enzyme.
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PMID:DNA-dependent RNA polymerase from Pseudomonas BAL-31. I. Purification and properties of the enzyme. 112 Jan 4

DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).
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PMID:Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III. 112 92

The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E. coli RNA polymerase by about 100 and 40 fold respectively. Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold. Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions. PDS inhibits transcription of deproteinized chromatin. Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase. Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked. If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS. These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin.
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PMID:Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides. 114 63

An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.
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PMID:A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. 118 7

A non-histone protein has been isolated from Ehrlich ascites tumor chromatin. The minimum molecular weight of this non-histone protein, estimated by sodium dodecyl sulfate gel electrophoresis and amino acid analysis, is approximately 10 to 11,000. This non-histone protein is acidic, contains 2.7% alkalilabile phosphorus, binds to DNA, and inhibits transcription of DNA in vitro by the homologous RNA polymerase. The per cent inhibition of RNA synthesis is not affected by increasing amounts of RNA polymerase, but is reduced by addition of excess DNA. In the presence of the non-histone protein, incorporation of [gamma-32P]ATP into RNA in the in vitro RNA synthesizing system is inhibited, with no apparent change in the average chain length of the RNA product. Inhibition of RNA synthesis is completely eliminated if the DNA template is allowed to interact with ATP prior to the addition of the non-histone protein. These results indicate that the observed repression of in vitro RNA synthesis is due to the effect of the non-histone protein on the DNA, inhibiting the initiation of RNA chain formation.
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PMID:Inhibition of transcription in vitro by a non-histone protein isolated from Ehrlich ascites tumor chromatin. 119 69


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