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)

Active eukaryotic RNA polymerase II (RNAP II) was purified by immunoaffinity chromatography, using a monoclonal antibody (mAb) that reacts with the highly conserved heptapeptide repeat of the largest subunit. This mAb (designated SWG16) was conjugated to CNBr-activated Sepharose and used to purify RNAP II from wheat germ and calf thymus. The subunit composition of the immunoaffinity-purified enzyme was essentially the same as RNAP II purified by conventional chromatography except that it contained only the form with the unproteolyzed largest subunit. Active enzyme could be eluted from the SWG16-Sepharose, at pH 7.9, with combinations of low molecular weight polyols and nonchaotropic salts. The superior eluting procedure used combinations of ethylene glycol (30-40%) and ammonium sulfate (0.5-0.75 M). Active enzyme also could be eluted with a synthetic peptide containing four repeats of the heptapeptide; however, the peptide was not as effective as the polyol and salt combinations for eluting the enzyme. This mAb should be useful for purifying RNAP II from many eukaryotic species. Because the elution of enzyme from the immunoadsorbent seems to be dependent upon the presence of a polyol, this antibody is referred to as a "polyol-responsive mAb." A procedure that helps to identify a polyol-responsive mAb and to optimize the eluting conditions is described. Polyol-responsive mAbs might have broad applicability to the purification of many labile enzymes by immunoaffinity chromatography.
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PMID:Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody. 232 14

RNA-dependent RNA polymerase activity was detected in both virion and nucleocapsid preparations of wheat rosette stunt virus, a plant rhabdovirus. The presence of nonionic detergent such as Nonidet P40 was essential for activity in reaction mixtures containing virions. The polymerase product was proved to be single-stranded RNA. By two-step controlled dissociation of the nucleocapsids, four subviral fractions (L protein, NS-N-RNA complex, NS protein, and N-RNA complex) were prepared. None of these fractions showed RNA polymerase activity when assayed individually. In experiments combining the various fractions, RNA synthesis was observed only when the L and NS proteins and the N-RNA complex were present in reaction mixtures.
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PMID:In vitro studies on the nucleocapsid-associated RNA polymerase of wheat rosette stunt virus. 318 29

A bacteriophage-coded RNA polymerase was isolated from bacteriophage-Xp10-infected Xanthomonas campestris pv. oryzae. The enzyme was purified to homogeneity through precipitation by poly(ethylene glycol) and chromatography on DEAE-cellulose, heparin--Sepharose 4B and blue-dextran--Sepharose 4B. It is composed of a single polypeptide of Mr96,000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA, host bacterium DNA and poly[d(A-T)] as templates. The optimal concentration of MgCl2 is 16 mM. The optimal temperature and pH are 37 degrees C and 8.0, respectively. The Km of ATP is 26 microM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xp10 RNA polymerase activity was retained. The enzyme activity was inhibited by KCl, spermidine, actinomycin D, heparin, blue dextran and ethidium bromide; it was resistant to rifampicin and streptovaricin. N-Ethylmaleimide did not affect the enzyme activity. The transcription site and product of Xp10 RNA polymerase upon Xp10 DNA were analyzed by DNA/RNA hybridization and polyacrylamide-agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xp10 genome and produce two RNA bands.
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PMID:Characterization of phage-Xp10-coded RNA polymerase. 372 Jul 43

RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.
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PMID:RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages. 615 35

We have further characterised a fraction of polynucleosomal chromatin from mouse cells which is enriched in transcribing regions, after separation by multistep partition in a dextran /poly(ethylene glycol) two-phase mixture [Faber, A. J. (1972) Methods Cell Biol. 16, 447--457]. This fraction contains an average of 16% of the total DNA and 63% of the 3-min pulse-labelled RNA in chromatin which, by a number of criteria, represents nascent RNA transcripts. Of the total transcribing RNA polymerase B molecules in chromatin, 52% are found in this fraction by titration with radioactive alpha-amanitin. About 28% of the single-copy DNA sequences in this fraction hybridise to nuclear polyadenylated RNA, compared with only 1.5% of the single-copy sequences in the remaining (nontranscribing) chromatin fraction.
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PMID:Characterisation of a chromatin fraction bearing pulse-labelled RNA. 1. Nascent RNA, RNA polymerase B and transcribed DNA sequence content. 617 75

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-N-N'-tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP(1) and VP(2) in addition to the proteins found associated with the viral minichromosome, i.e., VP(3) and histones H(2)A, H(2)B, H(3), and H(4). Our results suggest that the association of VP(1), VP(2), or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the "early" SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP(1) or VP(2) or both enhances the transcriptional activity and suggest that these "late" viral proteins may play a role in the regulation of expression of the SV40 genome.
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PMID:Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions. 625 78

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.
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PMID:Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution. 752 79

Leishmania RNA virus 1 produces a short viral RNA transcript corresponding to the 5' end of positive-sense single-stranded RNAs both in virally infected cells and in in vitro polymerase assays. We hypothesized that this short transcript was generated via cleavage of full-length positive-sense single-stranded RNA. A putative cleavage site was mapped by primer extension analysis to nucleotide 320 of the viral genome. To address the hypothesis that the short transcript is generated via cleavage at this site, two substrate RNAs that possessed viral sequence encompassing the putative cleavage site were created. When incubated with sucrose-purified viral particles, these substrate RNAs were site-specifically cleaved. The cleavage site of the in vitro-processed RNAs also mapped to viral nucleotide 320. The short-transcript-generating activity could be specifically abolished by proteinase K treatment of sucrose-purified viral particles and high concentrations of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], suggesting that the activity requires a proteinaceous factor and possibly intact viral particles. The cleavage activity is directly associated with short-transcript-generating activity, since only viral particle preparations which were capable of generating the short transcript in polymerase assays were also active in the cleavage assay. Furthermore, the short-transcript-generating activity is independent of the viral polymerase's transcriptase and replicase activities. We present a working model whereby cleavage of Leishmaniavirus RNA transcripts functions in the maintenance of a low-level persistent infection.
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PMID:The short transcript of Leishmania RNA virus is generated by RNA cleavage. 774 92

We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system.
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PMID:A highly efficient cell-free protein synthesis system from Escherichia coli. 877 39

We give a detailed account on the enzymatic synthesis of RNA conjugates by T7 RNA polymerase using modified initiator nucleotides during transcription. Following two different routes, ternary conjugates of guanosine-5'-monophosphate, poly(ethylene glycol), and anthracene were synthesized via phosphoramidite intermediates and characterized by a variety of spectroscopic techniques. Up to a degree of polymerization nPEG of about 17, these conjugates were efficiently incorporated into RNA by T7 RNA polymerase at the 5'-termini, thereby giving access to RNA conjugates required for biochemical studies as well as for the exploration of the catalytic potential of ribonucleic acids. The resulting conjugates are intact and functional.
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PMID:Ternary conjugates of guanosine monophosphate as initiator nucleotides for the enzymatic synthesis of 5'-modified RNAs. 1034 66


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