The coordinated activation and expression of genes by RNA polymerase II is an essential biological process for all eukaryotic cells.  Central to this process is recruitment of the general transcription machinery into a preinitiation complex (PIC) at the core promoter by gene-specific activator proteins.  The mediator coactivator complex plays an important role by serving as a bridge between upstream gene-specific activator proteins and the PIC (see diagram below).  Using the genetically tractable model organism Saccharomyces cerevisiae, our current research examines the hypothesis that mediator directly interacts with PIC components and stimulates RNA polymerase II activation through the general transcription factor TFIIH.  To address these issues, our research currently has two specific focuses:

1.  Mapping global protein interactions between mediator and PIC components.  Fluorescence resonance energy transfer (FRET) provides a powerful approach to analyze large, dynamic protein interaction networks.  FRET is performed on intact complexes (not isolated subunits) and is carried out in vivo in living cells.  We currently apply FRET technology to identify protein-protein interactions between subunits of mediator and components of the PIC.

2.  Identifying mediator subunits necessary for TFIIH-mediated activation of RNA polymerase II.  Mediator is an important coactivator for transcription by RNA polymerase II.  Recent reports suggest a critical function of mediator is to stimulate phosphorylation of the RNA polymerase II C-terminal domain (CTD).  This phosphorylation event is mediated by Kin28, a component of TFIIH, and results in an active RNA polymerase II.  We are employing classical genetic approaches and chromatin immunoprecipitation experiments to identify those mediator subunits necessary for stimulating Kin28 kinase activity and activation of RNA polymerase II.