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.
A photoactive nucleotide analogue of UTP, 5-azido-uridine 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 Km for the azido analogue was 27 microM and that of the natural substrate, UTP, was 7 microM. Photolysis of [gamma-32P]5-N3UTP in the presence of VSV transcription complexes resulted in selective radio-labelling of the L protein. This photolabelling was saturable with an apparent Kd of 28 microM. The L protein was protected from [gamma-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.
Hammond
D C
DC
School of Biological Sciences, Lucille P. Markey Cancer Center, University of Kentucky, Lexington 40506-0225.
Evans
R K
RK
Lesnaw
J A
JA
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D012333:RNA, Messenger / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D012333:RNA, Messenger; D014764:Viral Proteins; C072279:5-azidouridine-5'-triphosphate; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate