Mediator and TFIIH govern carboxyl-terminal domain-dependent transcription in yeast extracts

Abstract

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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.