Abstract
Transcript initiation by RNA polymerase II (pol II) requires a helicase within TFIIH to generate the unpaired template strand. However, pol II preinitiation complexes (PICs) lose the ability to synthesize RNA very rapidly upon exposure to ATP alone in the absence of other NTPs. This inactivation is not caused by the TFIIH kinase activity, the loss of transcription factors or pol II from the PIC, or the collapse of the initially formed transcription bubble. TFIIE is necessary for PIC formation, but TFIIE is not retained as a stable component in PICs prepared by our protocol. Nevertheless, activity can be at least partially restored to ATP-treated PICs by the readdition of TFIIE. PICs formed on premelted (bubble) templates require TFIIH for effective transcript elongation to +20. Incubation of bubble template PICs with ATP caused reduced yields of 20-mers, but this effect was partially reversed by the addition of TFIIE. Our results suggest that once the open complex is formed, TFIIH decays into an inactive configuration in the absence of nucleotides for transcription. Although TFIIE does not play a role in transcript initiation itself, inactivation resulting from ATP preincubation can be reversed by a remodeling process mediated by TFIIE. Finally, we have also uncovered a major role for TFIIF in the earliest stages of transcript elongation that is unique to bubble templates.