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J. Biol. Chem., Vol. 279, Issue 48, 49973-49981, November 26, 2004

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Purification of Active TFIID from Saccharomyces cerevisiae

EXTENSIVE PROMOTER CONTACTS AND CO-ACTIVATOR FUNCTION^*

Roy Auty, Hanno Steen¶, Lawrence C. Myers||, Jim Persinger**, Blaine Bartholomew**, Steven P. Gygi¶, and Stephen Buratowski

From the Department of Biological Chemistry and Molecular Pharmacology and the ^¶Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, the ^||Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, and the ^**Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901

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 affinity tag. Analysis of the final elution by mass spectrometry 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 ubiquitination 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 complexes can be analyzed by native gel electrophoresis. Phenanthroline-copper footprinting and photoaffinity cross-linking indicate that TFIID makes extensive contacts upstream and downstream of the TATA box. TFIID supports basal transcription and activated transcription, both of which are enhanced by TFIIA.

Received for publication, August 26, 2004 , and in revised form, September 20, 2004.

^* This work was supported in part by National Institutes of Health Grants GM62483 (to L. C. M.) and GM46498 (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Howard Hughes Medical Institute predoctoral fellowship.

Scholar of the Leukemia and Lymphoma Society. To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0696; Fax: 617-738-0516; E-mail: steveb{at}hms.harvard.edu.

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