A ROLE FOR ATP AND TFIIH IN ACTIVATION OF THE RNA-POLYMERASE-II PREINITIATION COMPLEX PRIOR TO TRANSCRIPTION INITIATION

A requirement for an ATP cofactor in synthesis of the first 8-10 bonds of promoter-specific transcripts by RNA polymerase II is well establi shed. Whether ATP is required for synthesis of the first phosphodieste r bond or at a slightly later stage in synthesis of nascent transcript s, however, remains controversial. Goodrich and Tjian (Goodrich, J. A. , and Tjian, R, (1994) Cell 77, 145-156) recently proposed that synthe sis of the first phosphodiester bond of promoter-specific transcripts by RNA polymerase II is independent of ATP and general transcription f actors TFIIE and TFIIH. Here we investigate this model, Taken together , our findings indicate that ATP, TFIIE, and TFIIH can have a profound effect on the efficiency of transcription initiation, First, we obser ve that synthesis of the first phosphodiester bond of transcripts init iated at the adenovirus 2 major late promoter depends strongly on ATP, TFIIE, and TFIIH in a transcription system reconstituted with RNA pol ymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and TFIIF. Secon d, we demonstrate that, in this enzyme system, ATP-dependent activatio n of transcription initiation can occur immediately prior to synthesis of the first phosphodiester bond of nascent transcripts, Finally, we demonstrate that the activated initiation complex is unstable and deca ys rapidly to an inactive state in the presence of the inhibitor ATP g amma S (adenosine 5'-O-(thio)triphosphate), even during reiterative sy nthesis of abortive transcripts.