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Molecular and Cellular Biology, July 2002, p. 4723-4738, Vol. 22, No. 13
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.13.4723-4738.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Steven L. Sanders,1,{dagger} Jennifer Jennings,2 Adrian Canutescu,2 Andrew J. Link,2 and P. Anthony Weil1*

Department of Molecular Physiology and Biophysics,1 Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-06152

Received 8 January 2002/ Returned for modification 13 February 2002/ Accepted 27 March 2002

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.

* Corresponding author. Mailing address: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. Phone: (615) 322-7007. Fax: (615) 322-7236. E-mail: tony.weil{at}mcmail.vanderbilt.edu.

{dagger} Present address: Wellcome/CR, UK Institute and Department of Pathology, University of Cambridge, Cambridge CB2 1QR, United Kingdom.

Molecular and Cellular Biology, July 2002, p. 4723-4738, Vol. 22, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.13.4723-4738.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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