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) promoter was studied with purified basal transcription factors, it was observed that transcription from negatively-supercoiled ML templates did not require transcription factor IIH (TFIIH). Addition of the basal factor TFIIE was highly stimulatory, but not absolutely required for this reaction. In contrast, transcription from relaxed or linear ML templates required both TFIIE and TFIIH. Adenylylimidodiphosphate (AMP-PNP), an ATP analog with a non-hydrolyzable beta-gamma phosphoanhydride bond, could support RNA synthesis from supercoiled templates, but not from 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 ATP-dependent step in transcription initiation by RNA polymerase II is caused by a requirement for the ATP-dependent helicase activity of the basal factor TFIIH. The experiments also show that transcription initiation by RNA polymerase II does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP per se.