Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTP.
8-Azido GTP (8-N3 GTP) was demonstrated to be polymerized into RNA by influenza virus-associated RNA polymerase at about one tenth the rate of GTP incorporation. The Km value for the azido analogue of GTP in primer-dependent RNA synthesis was 94 microM whereas Km for the natural substrate, GTP, was 6.7 microM. Upon exposure of a mixture of 8-N3 [alpha-32P]GTP and influenza virus ribonucleoprotein (RNP) complexes to ultraviolet light, the PB1 subunit of viral RNA polymerase was selectively radio-labeled. The photo-labeling of PB1 was competed strongly by GTP and to lesser extents by other nucleoside 5'-triphosphates. These results altogether support the prediction that the substrate-binding site (S site) of influenza RNA polymerase is located on the PB1 protein. In the presence of ApG primer, the 8-N3 GTP binding was reduced to about 40% level, suggesting that the GTP analogue can bind not only to the S site but also to the primer- and product-binding site (P site).
Asano
Y
Y
Department of Molecular Genetics, National Institute of Genetics, Shizuoka.
Mizumoto
K
K
Maruyama
T
T
Ishihama
A
A
D000345:Affinity Labels; D001386:Azides; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D009980:Influenza A virus / Q000201:enzymology*; D009711:Nucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D010777:Photochemistry
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D009711:Nucleotides; D010446:Peptide Fragments; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases