Probes of eukaryotic DNA-dependent RNA polymerase II-II: Covalent binding of two purine nucleoside dialdehydes to the initiation subsite

The catalytic center of wheat germ DNA-dependent RNA polymerase RNA polymerase II (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) as a model eukaryotic enzyme system was probed with two purine nucleoside dialdehydes, 6-methylthioinosinedicarboxaldehyde (MMPR-OP) and a derivative 6-(acetylaminoethyl)-1-naphthylamine-5-sulfonyl]thioinosinedicarboxaldehyde (AMPR-OP). Both drugs gave noncompetitive inhibition with respect to ³H]UMP incorporations into RNA, and inhibitor bindings were reversed with initiation substrates. The K_i values for MMPR-OP and AMPR-OP were determined to be 0.64 mM and 1.0 μM respectively. The drugs were covalently bound to the catalytic center by NaBH₄ reduction. Both were found bound to the largest enzyme subunit, IIa. It is essential lysine in the initiation subsite of the catalytic center located on the IIa subunit.