DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator

Abstract

Basal transcription factor TFIID comprises the TATA-box-binding protein, TBP, and associated factors, the TAF(II)s. Previous studies have implicated TAF(II)250 and TAF(II)150 in core promoter selectivity of RNA polymerase II. Here, we have used a random DNA binding site selection procedure to identify target sequences for these TAFs. Individually, neither TAF(II)250 nor TAF(II)150 singles out a clearly constrained DNA sequence. However, a TAF(II)250-TAF(II)150 complex selects sequences that match the Initiator (Inr) consensus. When in a trimeric complex with TBP, these TAFs select Inr sequences at the appropriate distance from the TATA-box. Point mutations that inhibit binding of the TAF(II)250-TAF(II)150 complex also impair Inr function in reconstituted basal transcription reactions, underscoring the functional relevance of Inr recognition by TAFs. Surprisingly, the precise DNA sequence at the start site of transcription influences transcriptional regulation by the upstream activator Sp1. Finally, we found that TAF(II)150 specifically binds to four-way junction DNA, suggesting that promoter binding by TFIID may involve recognition of DNA structure as well as primary sequence. Taken together, our results establish that TAF(II)250 and TAF(II)150 bind the Inr directly and that Inr recognition can determine the responsiveness of a promoter to an activator.