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Molecular and Cellular Biology, April 2002, p. 2842-2852, Vol. 22, No. 8
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.8.2842-2852.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Requirement of TRAP/Mediator for Both Activator-Independent and Activator-Dependent Transcription in Conjunction with TFIID-Associated TAFIIs

Hwa Jin Baek,1 Sohail Malik,1 Jun Qin,2 and Robert G. Roeder1*

Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021,1 Department of Biochemistry and Department of Cell Biology, Baylor College of Medicine, Houston, Texas 770302

Received 15 October 2001/ Returned for modification 26 November 2001/ Accepted 8 January 2002

The multiprotein human TRAP/Mediator complex, which is phylogenetically related to the yeast SRB/Mediator coactivator, facilitates activation through a wide variety of transcriptional activators. However, it remains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified a previously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific. An antibody that is specific for TRAP25 allowed quantitative immunodepletion of essentially all TRAP/Mediator components from HeLa nuclear extract, without detectably affecting levels of RNA polymerase II and corresponding general transcription factors. Surprisingly, the TRAP/Mediator-depleted nuclear extract displayed severely reduced levels of both basal and activator-dependent transcription from DNA templates. Both activities were efficiently restored upon readdition of purified TRAP/Mediator. Moreover, restoration of basal and activator-dependent transcription to extracts that were simultaneously depleted of TRAP/Mediator and TFIID (TBP plus the major TAFIIs) required addition of both TBP and associated TAFIIs, as well as TRAP/Mediator. These observations indicate that TAFIIs and Mediator are jointly required for both basal and activated transcription in the context of a more physiological complement of nuclear proteins. We propose a close mechanistic linkage between these components that most likely operates at the level of combined effects on the general transcription machinery and, in addition, a direct role for Mediator in relaying activation signals to this machinery.

* Corresponding author. Mailing address: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021. Phone: (212) 327-7600. Fax: (212) 327-7949. E-mail: roeder{at}rockvax.rockefeller.edu.

Molecular and Cellular Biology, April 2002, p. 2842-2852, Vol. 22, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.8.2842-2852.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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