title: Inactivation of the RNA polymerase of vesicular stomatitis virus by N-ethylmaleimide and protection by nucleoside triphosphates. Evidence for a second ATP binding site on L protein.
abstract: The purified RNA polymerase complex of vesicular stomatitis virus required added thiols for maximal activity, whereas polymerase activity from whole disrupted virions did not. Maximal activity of the purified polymerase complex required greater than or equal to 1 mM added dithiothreitol. The polymerase was inactivated by N-ethylmaleimide (NEM) at 0 degree C, with k2 = 528 +/- 26 M-1 min-1. Activity was recovered by addition of L protein, but not N or NS, to the NEM-inactivated complex, indicating that the NEM-sensitive group was present on the L protein. Nucleoside triphosphates protected the enzyme against inactivation by N-ethylmaleimide. ATP was most effective, with KD = 0.58 +/- 0.07 mM, a value close to the Km of ATP reported previously for initiation of RNA synthesis. dATP was nearly as effective, and GTP was slightly less effective than ATP. Non-hydrolyzable analogs of ATP protected weakly, whereas ADP and pyrimidine triphosphates gave very poor, but still measurable, protection. The ATP binding site thus identified differs from the protein kinase-associated ATP binding site identified on L protein by Sanchez et al. (Sanchez, A., De, B.P., and Banerjee, A. K. (1985) J. Gen. Virol. 66, 1025-1036) in having a substantially lower affinity for ATP. Two putative ATP binding sites were identified in the L protein amino acid sequence, but none were found in the N or NS sequences.
mesh_terms: D000818:Animals; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D012265:Ribonucleotides / Q000494:pharmacology*; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology*
chemical_list: D012265:Ribonucleotides; D012321:DNA-Directed RNA Polymerases; D005033:Ethylmaleimide; D004229:Dithiothreitol