Characterization of the guanosine-3'-diphosphate-5'-diphosphate binding site on E. coli RNA polymerase using a photoprobe, 8-azidoguanosine-3'-5'-bisphosphate.

Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoaffinity labeling with a GTP analog [gamma-32P]-8-AzidoGTP and a guanosine-3'-diphosphate-5'-diphosphate analog, 8-Azidoguanosine-3'-phosphate-5'-85'-32P]phosphate. The guanosine diphosphate photoprobe labeled the beta, beta', and sigma subunits with the sigma subunit being most heavily labeled. The GTP photoprobe also labeled the beta, beta', sigma subunits but the beta' subunit was most heavily labeled. In competition experiments guanosine-3'-diphosphate-5'-diphosphate decreased photolabeling by 8-Azidoguanosine-3'-phosphate-5'-[5'-32P]phosphate better than GTP, while the opposite was true for photolabeling with [gamma-32P]8- AzidoGTP. The guanosine diphosphate photoprobe inhibited transcription on E. coli DNA with Ki of ca. 150 microM. Present studies suggest a unique ppGpp binding site distinct from substrate binding site(s) and this photoprobe may be used to localize this binding site(s).