ATP activates transcription initiation from promoters by RNA polymerase II in a reversible step prior to RNA synthesis

Abstract

We have investigated the role that ATP plays in the synthesis of accurately initiated transcripts from the adenovirus 2 major late and mouse interleukin-3 promoters by a purified RNA polymerase II transcription system prepared from rat liver. The synthesis of 250-330 nucleotide run-off transcripts and 4-9 nucleotide Sarkosyl-resistant transcription intermediates requires ATP both for RNA synthesis and for activation of the system prior to RNA synthesis. Activation specifically requires an adenine nucleoside triphosphate containing a hydrolyzable beta, gamma-phosphoanhydride bond. ATP, adenine-9-beta-D-arabinofuranoside (araATP), and dATP are potent activators of transcription; they activate transcription to 50% of maximum at 2 microM. ATP analogs containing nonhydrolyzable beta, gamma-phosphoanhydride bonds such as adenyl-5'-yl imidodephosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and adenosine 5'-O-(thio)triphosphate (ATP gamma S) function efficiently in chain elongation, but do not activate transcription. Furthermore, ATP gamma S is a potent, reversible inhibitor of ATP activation. 20 microM ATP gamma S inhibits the synthesis of both full-length run-off transcripts and sarkosyl-resistant intermediates by 50% when the concentration of ATP is 10 microM. ATP gamma S inhibition can be overcome by high concentrations of ATP, dATP, araATP, or ddATP. Inhibition of the synthesis of Sarkosyl-resistant transcription intermediates by ATP gamma S is prevented by preincubation of the transcription enzymes and DNA template with ATP and magnesium prior to the addition of ATP gamma S and the remaining ribonucleoside triphosphates. Thus we argue that ATP activates the transcription system in a step prior to RNA synthesis.