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.
Highlights
After template melting generates the open complex, RNA polymerase II preinitiation complexes become inactive without transcription substrates
Exposure of Pol II Preinitiation Complexes to ATP Results in Their Rapid Inactivation for RNA Synthesis—Earlier studies indicated that the polymerase II (pol II) preinitiation complexes (PICs) is destabilized upon template melting [18, 19]
In the course of our recent investigations of the composition of minimal pol II PICs assembled on doublestranded templates [22], we noted that TFIIB, TFIIF, and TBP are all fully retained 5 min after an energy source (ATP or dATP) is supplied to generate the open complex
Summary
After template melting generates the open complex, RNA polymerase II preinitiation complexes become inactive without transcription substrates. Transcript initiation by RNA polymerase II (pol II) requires a helicase within TFIIH to generate the unpaired template strand. In the canonical path for assembly of the pol II3 PIC at promoters containing a TATA element, TBP (or TFIID) and TFIIB initially associate with the template, followed by TFIIF and pol II and by TFIIE and TFIIH [1]. The newly formed open complex is functionally unstable (i.e. if NTPs are not quickly supplied, the transcription complex becomes inactive) [18, 19] We investigate this phenomenon in the present paper and show that inactivation from ATP preincubation does not result from the loss of TFIIH or other factors from the PIC, nor is it caused by the kinase activity of TFIIH.
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