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The L protein of vesicular stomatitis virus transcription complexes is specifically photolabelled by 5-azido-uridine 5'-triphosphate, an analogue of the RNA polymerase substrate uridine 5'-triphosphate

Robert K. Evans

School of Biological Sciences, Lucille P. Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40506-0225, U.S.A.

A photoactive nucleotide analogue of UTP, 5-azidouridine 5'-triphosphate (5-N₃UTP), has been demonstrated to interact with the RNA polymerase of the vesicular stomatitis virus (VSV) transcription complex. Kinetic studies indicated that 5-N₃UTP served as an efficient replacement for UTP in in vitro polymerase reactions. The K_m for the azido analogue was 27 µM and that of the natural substrate, UTP, was 7 µM. Photolysis of [-³²P]5-N₃UTP in the presence of VSV transcription complexes resulted in selective radiolabelling of the L protein. This photolabelling was saturable with an apparent K_d of 28 µM. The L protein was protected from [-³²P]5-N₃UTP-mediated photolabelling by competing natural substrates (UTP, CTP, ATP, GTP). The stoichiometry of photoprobe incorporation into the transcription complex was close to unity with respect to the L protein. These data provide evidence that the nucleotide-binding domain of the VSV RNA polymerase contains amino acid residues of the L protein.

Present address: Department of Chemistry, Williams College, Williamstown, Massachusetts 01267, U.S.A.

Received 10 May 1991; accepted 30 September 1991.