Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB

Assembly and activity of yeast RNA polymerase II (Pol II) preinitiation com plexes (PIC) was investigated with an immobilized promoter assay and extrac ts made from wild-type cells and from cells containing conditional mutation s in components of the Pol II machinery. We describe the following findings : (1) In one step, TFIID and TFIIA assemble at the promoter independently o f holoenzyme. In another step, holoenzyme is recruited to the promoter. Mut ations in the CTD of pol II, Srb2, Srb4, and Srb5, and two mutations in TFI IB disrupt recruitment of all holoenzyme components tested without affectin g TFIID and TFIIA recruitment. These results indicate that the stepwise ass embly pathway is blocked after TFIID/TFIIA binding. (2) Both the Gal4-AH an d Gal4-VP16 activators stimulate formation of active PICs by increasing the extent of PIC formation. The Gal4-AH activator stimulated PIC formation by enhancing the binding of TFIID and TFIIA, whereas Gal4-VP16 could enhance the recruitment of TFIID, TFIIA, and holoenzyme. (3) Extracts deficient in TFIIA activity showed reduced assembly of all PIC components. These and oth er results suggest that TFIIA acts at an early step by enhancing the stable recruitment of TFIID. (4) An extract containing the TFIIB mutant E62G, had no defect in PIC formation, but had a severe defect in transcription. Simi larly, mutation of the TATA box reduced PIC formation only two- to fourfold , but severely compromised transcription. These results demonstate an invol vement of TFIIB and the TATA box in one or more steps after recruitment of factors to the promoter.