Proteomics of the Eukaryotic Transcription Machinery: Identification of Proteins Associated with Components of Yeast TFIID by Multidimensional Mass Spectrometry

Abstract

The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). Although TFIID constituents are required for transcription initiation of most mRNA encoding genes, the mechanism of TFIID action remains unclear. To gain insight into TFIID function, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits. Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed against each subunit, and the constellation of TBP- and TAF-associated proteins was directly identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. A number of novel protein-protein associations were observed, and several were characterized in detail. These interactions include association between TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6p transactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferase complex, including a putative ubiquitin-specific protease component. Our results provide important new insights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing a complete proteomic interaction map of the eukaryotic transcription apparatus.

We are grateful to B. Cairns and B. Andrews for gifts of valuable reagents and to X. Shen and C. Wu both for communication of results regarding the composition of the INO80 complex prior to publication and for providing INO80-specific reagents. We are especially grateful to J. Blackford for help and advice with the statistical analyses of DALPC data. Finally, we thank all of the members of the Weil and Link laboratories for helpful discussions.

This work was supported by National Institutes of Health Grant GM52461 (P.A.W.) and by Vanderbilt-Ingram Cancer Support grant 5P30CA68485, HHMI, and Ingram Family gift (A.J.L.).