ABSTRACT Cellular differentiation, development and homeostasis depend on regulation of gene expression, which is largely focused on the DNA transcription initiation process. During transcription initiation, Mediator, a large multi-protein complex conserved throughout eukaryotes, conveys regulatory signals to RNA polymerase II (RNAPII), the enzyme responsible for transcription of all protein-coding genes. Mediator includes 25-30 different polypeptides (depending on the specific organism) organized into Head, Middle and Tail modules, plus a dissociable kinase module (CKM) that includes the Cdk8 kinase, the only catalytically-active Mediator subunit. Mediator conformational rearrangements that stabilize preinitiation complex (PIC) components have explained the effect of Mediator on basal transcription. However, conformational rearrangements alone are insufficient to explain the response of Mediator to transcription factors (TFs) that enables transcription activation and repression. Here we propose biochemical, functional and cryo-EM studies of mammalian Mediator (mMED) that build on our previous work and explore the significance of mMED’s antagonistic interaction with the CKM and with MED26, a metazoan-specific, dissociable mMED subunit closely linked to modulation of mMED–RNAPII interaction. The CKM and MED26 interact with Mediator around a Head-Middle module interface (the CTD-binding gap) where RNAPII interaction is initiated by binding of the carboxy- terminal domain of the largest RNAPII subunit (the CTD). CKM-bound (CKM-mMED) and MED26-bound (MED26-mMED) forms of mMED were independently identified by various research groups shortly after Mediator’s discovery and we propose to test a mMED activation mechanism based on CKM-mMED to MED26- mMED interconversion that we hypothesize controls mMED interaction with RNAPII and PIC formation. In Aim1 we will Investigate the connection between CKM – MED26 antagonism and Mediator activation. We posit that the crux of the mMED activation mechanism is control of the initial CTD-dependent mMED– RNAPII interaction by antagonistic effects of the CKM (limits RNAPII interaction) and MED26 (required for RNAPII interaction) at the CTD-binding gap. We will use in vitro and in vivo approaches including biochemical, functional and genomic analyses to understand modulation of mMED interaction with RNAPII and its effects on transcription initiation. These studies will test a proposed activation mechanism that would explain the significance of mMED subpopulations with opposite functional effects and test whether interconversion between mMED forms can explain mMED activation. In Aim 2 we will determine the structural underpinnings that enable regulation of Mediator-RNAPII interaction. We postulate that TF targeting of CKM-mMED and subsequent effects on MED26 and CTD interaction at the CTD-binding gap are enabled by mMED structural rearrangements or changes in mMED conformational dynamics. Structural analysis of well-defin...