Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite.
9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.
D000345:Affinity Labels / Q000138:chemical synthesis; D001665:Binding Sites / Q000187:drug effects; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D006861:Hydrogen Peroxide; D015122:Mercaptopurine; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D004229:Dithiothreitol