The PIC, which minimally consists of activators, Mediator, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and pol II, is responsible for regulating expression of all protein-coding genes within the entire human genome and is over 3.5 MDa in size. Mediator, at approximately 1.2 MDa, represents a dominant "sub-assembly" within the PIC and works to control PIC function in ways that are not well characterized. The structural features of Mediator (large cavities, extended shape) coupled with its large size (ca. 320 x 160 x 150 Å) provide an extensive surface for interaction with other proteins within the PIC. Not surprisingly, Mediator has been shown to physically and/or functionally interact with TFIID, TFIIH, and pol II which, together with Mediator itself, represent over 90% of the total molecular mass of the PIC. As a "master regulator" of transcription, Mediator interactions within the PIC likely play fundamental roles in regulating transcriptional activation. Our lab is using a variety of biochemical and biophysical methods to better define how Mediator works together with other PIC components to regulate transcript initiation.