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J. Biol. Chem., Vol. 280, Issue 40, 33739-33748, October 7, 2005

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Mediator and TFIIH Govern Carboxyl-terminal Domain-dependent Transcription in Yeast Extracts^*

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

In Saccharomyces cerevisiae, the RNA polymerase II (RNA Pol II) carboxyl-terminal domain (CTD) is required for viability, and truncation of the CTD results in promoter dependent transcriptional defects. A CTD-less RNA Pol II is unable to support transcription in yeast extracts, but basal transcription reactions reconstituted from highly purified general transcription factors are CTD-independent. To reconcile these two findings, we have taken a biochemical approach using yeast extracts and asked whether there is a factor in the cell that confers CTD-dependence upon transcription. By placing a cleavage site for the tobacco etch virus protease prior to the CTD, we have created a highly specific method for removing the CTD from RNA Pol II in yeast whole cell extracts. Using derivatives of this strain, we have analyzed the role of the Srb8-11 complex, Mediator, and TFIIH, in CTD-dependent basal transcription by either mutation or immunodepletion of their function. We have found that Mediator is a direct intermediary of CTD-dependent basal transcription in extracts and that the requirement for Mediator and the CTD in basal transcription originates from their ability to compensate for a limiting amount of TFIIH activity in extracts.

Received for publication, June 3, 2005

^* This research was supported by National Institutes of Health Grant GM62483 (to L. C. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I.

¹ To whom correspondence should be addressed. Tel.: 603-650-1198; Fax: 603-650-1128;E-mail: larry.myers{at}dartmouth.edu.

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