TRANSCRIPTION INITIATION BY RNA-POLYMERASE-II DOES NOT REQUIRE HYDROLYSIS OF THE BETA-GAMMA PHOSPHOANHYDRIDE BOND OF ATP

When transcription by RNA polymerase II from the major-late (ML) promo ter was studied with purified basal transcription factors, it was obse rved that transcription from negatively-supercoiled ML templates did n ot require transcription factor IIH (TFIIH). Addition of the basal fac tor TFIIE was highly stimulatory, but not absolutely required for this reaction. In contrast, transcription from relaxed or linear ML templa tes required both TFIIE and TFIIH. Adenylylimidodiphosphate (AMP-PNP), an ATP analog with a non-hydrolyzable beta-gamma phosphoanhydride bon d, could support RNA synthesis from supercoiled templates, but not fro m linear templates. Since AMP-PNP cannot act as a cofactor for the DNA helicase activity of TFIIH, this finding independently supported the conclusion that TFIIH is not required for transcription of negatively- supercoiled templates. Taken together, these data indicate that the AT P-dependent step in transcription initiation by RNA polymerase II is c aused by a requirement for the ATP-dependent helicase activity of the basal factor TFIIH. The experiments also show that transcription initi ation by RNA polymerase II does not require hydrolysis of the beta-gam ma phosphoanhydride bond of ATP per se.