Transcriptional activation often requires protein cofactors in addition to the general transcription machinery which includes TFIIB, TFIID, TFIIF, TFIIH and RNA polymerase II (pol II). At present, there are three general cofactors (TAF/IIs, USA, and Mediator) commonly thought to be critical for activator function. Although many studies have been conducted to identify the components and functional properties of these general cofactors, the relative contribution of each general cofactor in gene activation and the mechanism by which each cofactor function remain unclear. To address this important issue, we have developed a TAF/II- independent activation system reconstituted with only recombinant general transcription factors (TBP, TFIIB, TFIIE, and TFIIF), a recombinant cofactor (PC4), and epitope-lagged multi-protein complexes (TFIIH and pol II). In this system, PC4 is the only cofactor required for Gal4-VP16-mediated activation, thereby providing a unique in vitro functional assay to evaluate the role of each general cofactor in transcriptional activation. Since TAF/II-independent activation mediated by Gal4-VP16 can be recapitulated with only PC4 and components of the general transcription machinery, we hypothesize that some general transcription factors may play a role analogous that TFIIH can significantly affect activator function in our TAF/II-independent activation system and that TFIIH exhibit several activities also found in TFIID have prompted us to investigated whether TFIIH can indeed functionally substitute for TAF/IIs. Accordingly, we will perform order-of-addition and functional recruitment assays to address the role of general transcription factors and PC4 in TAF/II-independent activation. Although TAF/II-independent activation represents a new paradigm in eukaryotic gene regulation, many activators such as estrogen receptor (ER) strictly rely on TAF/IIs for activated transcription. Since a highly purified ER-dependent in vitro activation system has also been established in our laboratory, we will dissect the role of TAF/IIs in regulating the step of pre-initiation complex assembly and explore the hypothesis that ligand-dependent activation mediated by ER is likely due to the recruitment of Mediator which interacts with ER in a ligand- dependent manner. Collectively, these studies will shed light on the role and functional redundancy or transcriptional or transcriptional synergism of general cofactors in activator-dependent transcription.
