Abstract
Transcription activators trigger transcript production by RNA polymerase II (RNAPII) via the Mediator coactivator complex. Here, the dynamics of activator, Mediator, and RNAPII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNAPII to the template required an activator and an upstream activating sequence (UAS) but not a core promoter. While Mediator and RNAPII sometimes bind as a pre-formed complex, more commonly Mediator binds first and subsequently recruits RNAPII to form a pre-initiation complex precursor (pre-PIC) tethered to activators on the UAS. Interestingly, Mediator occupancy has a highly non-linear response to activator concentration, and fluorescence intensity measurements show that Mediator preferentially associates with templates having at least two activators bound. Statistical mechanical modeling suggests this "synergy" is not due to cooperative binding between activators but instead occurs when multiple DNA-bound activator molecules simultaneously interact with a single Mediator.