title: PIC Activation through Functional Interplay between Mediator and TFIIH.
abstract: The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address: maliks@rockefeller.edu.
authors.forename: Sohail
authors.initials: S
authors.lastname: Malik
authors.affiliation: Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Xue
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D056892:Mediator Complex / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D051758:Transcription Factor TFIIH / Q000378:metabolism*; D061785:Transcription Initiation, Genetic
chemical_list: D056892:Mediator Complex; D051758:Transcription Factor TFIIH
keywords: Mediator coactivator complex; RNA polymerase II; preinitiation complex; promoter melting; triptolide