Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry.
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.
Sanders
Steven L
SL
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Jennings
Jennifer
J
Canutescu
Adrian
A
Link
Andrew J
AJ
Weil
P Anthony
PA
D002853:Chromatography, Liquid / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism*; D007120:Immunochemistry / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D009154:Mutation; D020543:Proteome; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020543:Proteome; C118425:SAGA protein, Emericella nidulans; C075111:SWI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C116141:TAF9 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII