Purification of Active TFIID from Saccharomyces cerevisiae

Abstract

The basal transcription factor TFIID is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Although TBP alone binds to the TATA box of DNA and supports basal transcription, the TAFs have essential functions that remain poorly defined. In order to study its properties, TFIID was purified from Saccharomyces cerevisiae using a newly developed affin- ity tag. Analysis of the final elution by mass spectrome- try confirms the presence of all the known TAFs and TBP, as well as Rsp5, Bul1, Ubp3, Bre5, Cka1, and Cka2. Both Taf1 and Taf5 are ubiquitinated, and the ubiquiti- nation pattern of TFIID changes when BUL1 or BRE5 is deleted. Purified TFIID binds specifically to promoter DNA in a manner stabilized by TFIIA, and these com- plexes can be analyzed by native gel electrophoresis. Phenanthroline-copper footprinting and photoaffinity cross-linking indicate that TFIID makes extensive con- tacts upstream and downstream of the TATA box. TFIID supports basal transcription and activated transcrip- tion, both of which are enhanced by TFIIA. TFIID is a large, multisubunit complex that is