THE GCN4P ACTIVATION DOMAIN INTERACTS SPECIFICALLY IN-VITRO WITH RNA-POLYMERASE-II HOLOENZYME, TFIID, AND THE ADAP-GCN5P COACTIVATOR COMPLEX

The Gcn4p activation domain contains seven clusters of hydrophobic res idues that make additive contributions to transcriptional activation i n vivo, We observed efficient binding of a glutathione S-transferase ( GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subuni ts of transcription factor IID (TFIID) (yeast TAF(II)20 [yTAF(II)20], yTAF(II)60, and yTAF(II)90), the holoenzyme mediator (Srb2p, Srb4p, an d Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobi c clusters in the Gcn4p activation domain. Alanine substitutions in si ngle clusters led to moderate reductions in binding, double-cluster su bstitutions generally led to greater reductions in binding than the co rresponding single-cluster mutations, and mutations in four or more cl usters reduced binding to all of the coactivator proteins to backgroun d levels. The additive effects of these mutations on binding of coacti vator proteins correlated with their cumulative effects on transcripti onal activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a c ardinal function of the Gcn4p activation domain, As judged by immunopr ecipitation analysis, components of the mediator were not associated w ith constituents of TFIID and Adap-Gcn5p in the extracts, implying tha t GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAF(II)90, and the yTAF(II)20, -60, and -90 proteins were coimm unoprecipitated with Ada3p, revealing a stable interaction between com ponents of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with T FIID via the Adap-Gcn5p complex or some other adapter proteins. The ab ility of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protei n can provide an explanation for the observation that yTAF(II) protein s are dispensable for activation by Gcn4p in vivo.