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Gazina EV Smidansky ED Holien JK Harrison DN Cromer BA Arnold JJ Parker MW Cameron CE Petrou S 《Journal of virology》2011,85(19):10364-10374
Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase. 相似文献
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Monica T. M. Shander Carlo Croce Roberto Weinmann 《Journal of cellular physiology》1982,113(2):324-328
A human fibrosarcoma cell line, HT-1080-6TG-9AM, resistant to α-amanitin at concentrations up to 10 μg/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled α-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50-to 100-fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to α-amanitin is due to an alteration of RNA polymerase II. 相似文献
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RNA polymerase II subunit composition, stoichiometry, and phosphorylation. 总被引:18,自引:7,他引:18
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RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme. 相似文献
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We have purified DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing approximately 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc. 相似文献
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Clones resistant to the cytotoxic action of alpha-amanitin have been isolated from a strain of fetal human lung diploid fibroblasts. Resistant clones were recovered at a frequencey of 5 X 10(-8) after single-step selections following mutagenesis with the mutagen ethyl methane sulfonate. Following propagation in drug-free medium, the clones retained the selected phenotype and in both growth and plating experiments showed a 10-50-fold higher resistance than wild-type cells to the cytotoxicity of 0.25 microgram/ml alpha-amanitin. The alpha-amanitin sensitivity of RNA polymerase II purified from the mutant cells suggests the presence of two forms of the enzyme, one similar to that found in wild-type cells and a second form with increased resistance to alpha-amanitin inhibition. These results are consistent with previous evidence that alpha-amanitin resistance behaves as a codominant marker in mammalian cells. 相似文献
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