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.
Nair
Dhanalakshmi
D
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Kim
Yeejin
Y
Myers
Lawrence C
LC
D010450:Endopeptidases; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D051758:Transcription Factor TFIIH / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II; D010450:Endopeptidases; C099488:TEV protease