Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme

Abstract

In eukaryotes, transcriptional activators have been proposed to function by recruiting the RNA polymerase II (Pol II) machinery^1,2,3, by altering the conformation of this machinery⁴,⁵, or by affecting steps after initiation^6,7,8, but the evidence is not definitive. Genomic footprinting of yeast TATA-box elements reveals activator-dependent alterations of chromatin structure⁹ and activator-independent protection¹⁰, but little is known about the association of specific components of the Pol II machinery with promoters in vivo. Here we analyse TATA-box-binding-protein (TBP) occupancy of 30 yeast promoters in vivo. We find that TBP association with promoters is stimulated by activators and inhibited by the Cyc8–Tup1 repressor, and that transcriptional activity correlates strongly with the degree of TBP occupancy. In a small subset of promoters, TBP occupancy is higher than expected when gene activity is low, and the activator-dependent increase is modest. TBP association depends on the PolII holoenzyme component Srb4, but not on the Kin28 subunit of the transcription factor TFIIH, even though both proteins aregenerally required for transcription. Thus in yeast cells, TBP association with promoters occurs in concert with the Pol II holoenzyme, activator-dependent recruitment of the Pol II machinery occurs at the vast majority of promoters, and Kin28 acts after the initial recruitment.