Mediator and TFIIH govern carboxyl-terminal domain-dependent transcription in yeast extracts.
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.
Mesh Terms:
Endopeptidases, Fungal Proteins, Gene Expression Regulation, Fungal, Promoter Regions, Genetic, RNA Polymerase II, Recombinant Proteins, Saccharomyces cerevisiae, Transcription Factor TFIIH, Transcription, Genetic
Endopeptidases, Fungal Proteins, Gene Expression Regulation, Fungal, Promoter Regions, Genetic, RNA Polymerase II, Recombinant Proteins, Saccharomyces cerevisiae, Transcription Factor TFIIH, Transcription, Genetic
J. Biol. Chem.
Date: Oct. 07, 2005
PubMed ID: 16076843
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