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

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 β, γ-phosphoanhydride bond. ATP, adenine-9-β-D-arabinofuranoside (araATP), and dATP are potent activators of transcription; they activate transcription to 50% of maximum at 2 μM. ATP analogs containing nonhydrolyzable β,γ-phosphoanhydride bonds such as adenyl-5'-yl imidodephosphate, adenosine 5'-(β,γ-methylene)triphosphate, and adenosine 5'-O-(thio)triphosphate (ATPγS) function efficiently in chain elongation, but do not activate transcription. Furthermore, ATPγS is a potent, reversible inhibitor of ATP activation, 20 μM ATPγS inhibits the synthesis of both full-length run-off transcripts and Sarkosyl-resistant intermediates by 50% when the concentration of ATP is 10 μM. ATPγS inhibition can be overcome by high concentrations of ATP, dATP, araATP, or ddATP. Inhibition of the synthesis of Sarkosyl-resistant transcription intermediates by ATPγS is prevented by preincubation of the transcription enzymes and DNA template with ATP and magnesium prior to the addition of ATPγS and the remaining ribonucleoside triphosphates. Thus we argue that ATP activates the transcription system in a step prior to RNA synthesis.