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)

Optimal conditions for isolation of rooster liver nuclei were studied for the purification of RNA polymerases I and II by varying the concentrations of sucrose, magnesium chloride and spermine in the isolation medium. Addition of spermine and/or magnesium chloride to the hypertonic sucrose medium was found to be advantageous for the purification. Heparin-Sepharose chromatography can be recommended for purification of RNA polymerases when used in a stepwise manner. Furthermore, gradient elution in the heparin-Sepharose chromatography was found to separate RNA polymerases I and II. RNA polymerase III was eluted with RNA polymerase I. A few minor peaks of RNA polymerase II activity were detected with gradient elution. Factors influencing the affinity of RNA polymerases towards heparin-Sepharose are discussed.
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PMID:RNA polymerases from avian liver. Isolation and fractionation by heparin-sepharose chromatography. 51 13

Spermatogenesis is a complex developmental process which sequentially generates several different germ cell types. These cell types from rainbow trout (Salmo gairdnerii) testis were separated by sedimentation in serum albumin gradients and characterized on the basis of their physical properties, chronological appearance, and protein synthesis. The rate of RNA synthesis, the types of RNA made, and the RNA polymerase activities present were determined for each cell type. The rate of RNA synthesis decreased from a high level in spermatogonia and spermatocytes to a low level in early spermatids and was absent in late spermatids and mature spermatozoa. Newly synthesized RNA in spermatogonia and spermatocytes consisted of a variety of molecular weight species, including 18 S and 28 S ribosomal RNAs. The synthesis of high molecular weight RNAs, especially ribosomal RNAs, decreased drastically in early spermatids, leading to the synthesis of only small molecular weight RNAs. RNA polymerase I and II were present in all cell types but the activities of both showed large decreases between spermatocytes and middle spermatids. Both RNA polymerase activities were almost absent from spermatozoa. The activities of RNA polymerase I and II from unfractionated testis cells at different stages of hormone-induced spermatogenesis were quantitated by fractionation of the solubilized extract on DEAE-cellulose. Both polymerases showed major decreases in activity which began near the chronological mid-point of development. For polymerase I the decrease in activity was over 400 fold, for polymerase II over 200 fold. The number of RNA polymerase II molecules per testis cell, quantitated by the binding of [3H]amanitin to cell extracts, also decreased markedly during spermatogenesis. The reduction in polymerase II activity was accompanied by a parallel 200-fold decrease in[3H]amanitin binding. The reduction in polymerase activity appears, therefore, to be due to an actual reduction in the cellular content of RNA polymerase II molecules. These results suggest that transcription in maturing testes is regulated, at least in part, by the concentrations of the RNA polymerases.
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PMID:RNA synthesis and RNA polymerase activities in germ cells of developing rainbow trout testis. 51 81

Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 x 10(4) molecules of RNA polymerases I and III (altogether) and 1 x 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 x 10(4)-2.5 x 10(4) form-I and -III enzyme molecules (altogether) and a further 7 x 10(3)-8 x 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.
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PMID:A comparison of methods for extracting ribonucleic acid polymerases from rat liver nuclei. 53 87

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.
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PMID:Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II. 55 98

Influenza virus polypeptides were not synthesized in wild-type CHO-S-infected cells in the presence of alpha-amanitin, but were synthesized in CHO-Amal cells, a mutant cell line whose DNA-dependent RNA polymerase II is specifically resistant to this drug, indicating that this cellular enzyme is involved in influenza virus replication. The results of experiments designed to detect viral polypeptides synthesized from primary transcripts suggest that the synthesis of a cellular RNA species by RNA polymerase II is required for primary transcription of the influenza virus genome.
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PMID:Synthesis of influenza virus polypeptides in cells resistant to alpha-amanitin: evidence for the involvement of cellular RNA polymerase II in virus replication. 56 Nov 96

When partially purified Ehrlich ascites tumor RNA polymerase II was further purified on a column of phosphocellulose, stimulation of its catalysis of RNA synthesis by stimulatory factor S-II was greatly decreased. This decrease in sensitivity to the stimulatory factor was reversible: the enzyme eluted from phosphocellulose became sensitive to the factor when mixed with a protein fraction eluted from the phosphocellulose at high salt concentration. Evidence was obtained that this protein, named helper protein, binds, to the enzyme eluted from phosphocellulose, causing it to recover sensitivity to stimulatory factor S-II.
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PMID:Protein which interacts with a stimulatory factor of RNA polymerase II of Ehrlich ascites tumor cells. 56 40

Class II DNA-dependent RNA polymerases were purified from soybean tissues of different physiological states: (1) from seed embryo tissue, representative of a quiescent, low metabolic state and (2) from auxin-treated hypocotyl tissue, representative of a highly proliferative and metabolically active state. Dodecyl sulfate, polyacrylamide gel electrophoresis indicates that RNA polymerase II from embryonic tissue consists largely (90-95%) of the form IIA enzyme, the largest subunit having a molecular weight of 215 000. RNA polymerase II from hypocotyl tissue is exclusively a form IIB enzyme, the largest subunit having a molecular weight of 180 000. Polypeptides common to RNA polymerases IIA and IIB have the following molecular weights: 138 000; 42 000; 27 000; 22 000; 19 000; 17 600; 17 000; 16 200; 16 100; and 14 000. Peptide mapping in the presence of dodecyl sulfate suggests that the 215 000 and 180 000 subunits possess similar peptide fragments. Plant embryo tissues do not contain protease activity capable of cleaving the 215 000 subunit to the 180 000 subunit, but proliferating plant tissues do contain such an activity. Mixing experiments indicate that appreciable amounts of RNA polymerase IIB are not being artifactually produced during protein purification.
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PMID:Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues. 56 12

Carefully controlled preparation of chromatin from purified chick liver nuclei yielded over 50% native chromatin as shown by the analysis of the nucleosome pattern after micrococcal nuclease digestion. The size of DNA in this chromatin as analyzed on alkaline sucrose gradients varied from 10S to 19S, the majority being 14S. All endogenous RNA polymerases were represented in the chromatin preparation although to different extents: RNA polymerase I was the most and RNA polymerase II the least abundant. Initiation studies showed that endogenous RNA polymerase II was capable of initiating RNA chains during 5 min. Saturation of chromatin with purified homologous RNA polymerase II increased initiation to 10 min. The addition of heparin caused the RNA transcribed to be larger in size and of increased yield. Chromatin transcription with added purified RNA polymerase II in the presence of heparin produced RNA as large as 32S. A chromatin preparation of this kind would therefore be suitable to transcribe any eukaryotic gene invitro provided additional homologous RNA polymerase II is used.Images
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PMID:Characterization of chick liver chromatin and analysis of its in vitro transcription products. 56 11

The DNA-dependent RNA polymerases II or B (ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from the mushroom Agaricus bisporus has been purified to apparent homogeneity. The purification procedures involve precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-cellulose chromatography, and exclusion chromatography on Bio-Gel A-1.5M. With this procedure 11 mg of RNA polymerase II is recovered from 1.5 kg of mushroom tissue. RNA polymerase II from Agaricus bisporus has 12 subunits with the following molecular weights: 182,000, 140,000, 89,000, 69,000, 53,000, 41,000, 37,000, 31,000, 29,000, 25,000, 19,000, and 16,500. Purified RNA polymerase II from Agaricus bisporous was half-maximally inhibited by the mushroom toxin alpha-amanitin at a concentration of 6.5 microgram/mL (7 X 10(-6) M), which is 650-fold more resistant than mammalian RNA polymerases II. The apparent Ki for the alpha-amanitin-RNA polymerase complex was estimated to be 12 X 10(-6) M. The activity of purified RNA polymerase II from the mushroom was quite typical of other eukaryotic RNA polymerase II with regard to template preference, salt optima, and divalent metal cation optima.
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PMID:Purification and characterization of RNA polymerase II resistant to alpha-amanitin from the mushroom Agaricus bisporus. 57 Apr 13

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at approximately 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCL2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undergraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated approximately 3 pmoles of [3H]UMP/microgram DNA at 25 C for 15 min, and approximately 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, alpha-amanitin (20 micrograms/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with alpha-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).
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PMID:RNA synthesis in isolated nuclei of the dinoflagellate Crypthecodinium cohnii. 57 93


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