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)

A highly selective affinity label was introduced into the T7 phage RNA polymerase by means of GMP ortho-formylphenyl ester and [alpha-32P]UTP nearby the enzyme's active site, which was located using limited cleavage technique. Hydroxylamine, bromine, N-chlorosuccinimide, and cyanogen bromide were employed as the reagents. Analysis of gel-electrophoretic patterns of the cleavage products led to a conclusion that Lys631 is the target of labelling. The region nearby this residue has a high degree of sequence homology with regions of RNA polymerases from T3 and SP6 phages and yeast mitochondria.
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PMID:[Localization of a lysine residue near the site of initiating substrate binding of T7 bacteriophage RNA polymerase]. 250 Sep 35

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [alpha-33P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerase. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.
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PMID:Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius. 250 28

A dot-hybridization assay using 32P-labelled RNA probes (+RNA and cRNA) transcribed from potato spindle tuber viroid (PSTV) cDNA was described. A complete cDNA copy of PSTV, originally cloned in pBR 322 (pAV 401) was subcloned in the BamHI site of a 'Riboprobe' cloning vectors pSP 64 and pSP 65 in opposite orientations. The reconstructed plasmids were designated pDX 1 and pDX 4, respectively. Transcription of pDX 1 and pDX 4 plasmids by SP6 RNA polymerase resulted in the generation of PSTV-specific RNA (+RNA) and PSTV complementary RNA (cRNA), respectively. The cRNA probe was much more sensitive than the +RNA probe and the nick-translated cDNA probe from the plasmid pAV 401 for the detection of PSTV in clarified plant sap. As little as 1.4 pg of purified PSTV mixed in clarified sap from uninoculated tomato leaves has been detected using cRNA probe. A relatively simple procedure using cetyltrimethyl ammonium bromide (CTAB) as nucleic acid precipitant and an enrichment step for the purification of PSTV was described.
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PMID:Use of [32P]RNA probes for the dot-hybridization detection of potato spindle tuber viroid. 302 43

Viruses isolated from the yeast Yarrowia lipolytica possess a DNA-independent RNA polymerase activity which is inhibited by ethidium bromide and by sodium pyrophosphate but not by actinomycin D. RNA synthesis is maximum at pH 8.0 and at 30 degrees C. Newly synthesized RNA molecules are largely released from the particles, are single-stranded and are able to hybridize with denatured viral genomic RNA.
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PMID:In vitro RNA synthesis by viruses isolated from the yeast Yarrowia lipolytica. 309 Oct 94

We have examined the effects of six DNA binding drugs upon initiation at the lac UV5 promoter by Escherichia coli RNA polymerase. Experiments were directed at determining the influence of added drug on open complex formation, open complex stability, initiation from the open complex, and stability of the resulting initiated complex. The narrow groove binding drugs distamycin and 4',6-diamidino-2-phenylindole dihydrochloride were more effective in inhibiting initiation through their effect on the first three of these factors than were the intercalators ethidium bromide, daunomycin, and actinomycin. The bisintercalator bis(daunomycin) inhibited open complex formation better than its parent daunomycin. With the possible exception of actinomycin, the drugs tested were not able to disrupt preformed initiated complex, in contrast to their destabilizing effect upon the open complex. Combined with other results, the data suggest that the antitumor activity of daunomycin is unlikely to result from its effect on transcription. We compare the relative effectiveness of the drugs with the known physical properties of the corresponding drug-DNA interactions. The rate of open complex formation seems to be influenced by both the on and off rates of the drug, probably due to the relative slowness of open complex formation. This is in contrast to elongation, a much quicker process, which seems to be limited by the drug off rate alone; these considerations may possibly rationalize the difference in relative effect of particular drugs upon initiation and elongation. All drugs were able actively to disrupt open complex, although to substantially different extents; some possible mechanisms for this disruption, and the insensitivity of the initiated complex, are discussed.
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PMID:Effect of drug-DNA interactions upon transcription initiation at the lac promoter. 329 37

E. coli RNA polymerase was selectively labelled in the presence of promoters at a histidine residue of the beta-subunit by treatment with GDP beta-imidazolide and then with [alpha-32P]UTP (or [alpha-33P]UTP). Partial cyanogen bromide cleavage of the labelled polypeptide afforded a series of "single-hit" labelled peptides, the electrophoretic pattern of which suggested that the labelling site was His1237. This conclusion was confirmed by a similar pattern obtained with products of the cyanogen bromide cleavage of a radioactive peptide obtained by the limited trypsinolysis (C-terminal peptide consisting of 423 amino acid residues). Interpretation of our earlier results in favour of His1116 as the labelling point (Dokl. Acad. nauk SSSR, 1985, v. 281, p. 723) was incorrect due to the electrophoretic "compression" of three labelled peptide bands.
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PMID:[Localization of a histidine residue in the binding site for the initiating substrate of E. coli RNA-polymerase]. 331 73

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

The [alpha-32P]3'-azido-3'-deoxyxylofuranosyl analogues of 5'-ATP and 5'-GTP were used as photoaffinity probes to derivatize two distinctly different active sites of the DNA-dependent RNA polymerase obtained from Escherichia coli. The sites derivatized by these probes are both located on the beta subunit. Radioactive 32P containing cyanogen bromide peptides produced from the derivatized beta subunits were observed to give either one of two distinct patterns by high pressure liquid chromatography or TLC analysis. These sites are used alternately for the synthesis of successive phosphodiester bonds. These results support our rotational translocation model for transcription.
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PMID:RNA polymerase. Direct evidence for two active sites involved in transcription. 388 32

Echinomycin, daunomycin, ethidium bromide, nogalamycin, chromomycin, mithramycin, and olivomycin inhibit RNA synthesis by RNA polymerase by interacting with the DNA template. Chromomycin and olivomycin form complexes with DNA, preferably in the helical form, but not with RNA. These complexes require guanine in DNA and the addition of a stoichiometric amount of bivalent cation. None of the other antibiotics requires the presence of any single base in the template for its action.
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PMID:Base specificity in the interaction of polynucleotides with antibiotic drugs. 2531 97

Ribonucleic acid (RNA) synthesis was examined in cold-shocked Bacillus subtilis cells. The cells were grown to mid-log stage, harvested, and cold shocked. RNA synthesis was monitored by the incorporation of [3H]uridine triphosphate or [alpha 32P]adenosine triphosphate into trichloroacetic acid-precipitable material in the presence of all four nucleoside triphosphates. The inhibition of RNA synthesis in cold-shocked cells by lipiarmycin, ethidium bromide, rifampin. or streptolydigin was analyzed using mutant or wild-type cells. Also examined were the effects of temperature, salt concentration, and the addition of polyamines or highly phosphorylated nucleotides. In ultraviolet-irradiated and cold-shocked cells, RNA wynthesis decreased to low levels. The addition of exogenous phi 29 or TSP-1 template to these cells caused a 13- to 20-fold increase in RNA synthesis, as monitored by trichloroacetic acid-precipitable counts. RNA synthesized in the presence of phi 29 deoxyribonucleic acid (DNA) hybridizes mainly to EcoRI fragments A and C of phi 29 DBA, These two fragments direct transcription by purified RNA polymerase in vitro and hybridize to early phi 29 DNA produced in vivo. Our results with TSP-1 DNA in this system indicated that the RNA produced hybridizes to the same fragments as early RNA produced in vivo. Plasmic pUB110 DNA was not transcribed in this system.
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PMID:Transcription of exogenous and endogenous deoxyribonucleic acid templates in cold-shocked Bacillus subtilis. 615 74


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