Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTP.

Abstract:

: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).

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Asano Y,Mizumoto K,Maruyama T,Ishihama A

doi

10.1093/oxfordjournals.jbchem.a124762

subject

Has Abstract

pub_date

1995-03-01 00:00:00

pages

677-82

issue

3

eissn

0021-924X

issn

1756-2651

journal_volume

117

pub_type

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