The mechanism of the mediator coactivator complex in transcription

The growth, differentiation and survival of eukaryotic organisms requires appropriate, regulated expression of thousands of genes. Control of the expression of protein coding genes involves extraordinary coordination between numerous transcription factors. While much is understood about the factors required for activated transcription in eukaryotic cells, there is a lack of knowledge in the field about the detailed mechanism of action of coactivator complexes. The work in this thesis offers an advance in our understanding of the mechanism of a recently characterized coactivator complex, the multisubunit mediator. Current models in the field suggest that mediator functions to recruit/stabilize formation of a preinitiation complex (PIC), which is composed of RNA Polymerase II and all of the general transcription factors. Consistent with this model, mediator and the general transcription factor TFIID interact cooperatively to stimulate each others recruitment to DNA. This occurs in either the presence or absence of an activator, offering an explanation for the observation that mediator stimulates both basal and activated transcription. The assembly of a complex of mediator, TFIID, and TFIIA (the DAMed complex) stimulates transcription in a manner that correlates with enhanced assembly of a PIC on promoter DNA. TFIID is composed of the TATA binding protein (TBP) and the TAF coactivators. All promoters require assembly of TBP for initation of transcription, TAFs are differentially required. Offering insight into the mechanism of activated transcription on TAF independent (TAFindep) promoters, mediator and TBP (which form a TAMed complex) also assemble cooperatively, stimulate transcription, and enhance PIC assembly. Intriguingly, the order of assembly of a DAMed and TAMed complex is critical for its function: pre-assembly of mediator prevents both TFIID and TBP binding, and inhibits PIC formation and transcription. Possibly analogous to the mechanism of transcriptional inhibition in mitosis, mediator that contains CDK8 can phosphorylate TBP (in the presence or absence of TAFs). This modification correlates with a decrease in PIC assembly and inhibition of activated transcription.