Mediator is an essential regulator of eukaryotic transcription that orchestrates transcription factors and RNA polymerase II (Pol II) assembly onto a gene promoter to facilitate transcription initiation. Mutations in several human Mediator subunits have been linked to neurological disorders and cancers, exemplifying the importance of proper Mediator function in human health. Yet, how Mediator orchestrates transcription is still a big black box. The structural complexity of Mediator has been a roadblock for structure-function studies that attempt to shed light on that black box. We have developed the experimental system that overcomes this roadblock by capturing and visualizing Mediator in action during transcription, thereby determining the transcription activation mechanisms by Mediator. Mediator from yeast is composed of 21 subunits with a molecular mass over 1 MDa. Despite of significant recent progress, the structure of the most biological relevant form of Mediator, when it is in the context of active transcription initiation (e.g. DNA-activator-Mediator has not been available because of two primary limitations: 1) lack of well-defined recombinant forms of the Mediator complex; and 2) lack of biochemical system to reconstitute the Mediator in active transcription form in vitro. To overcome these two limitations, we first developed a new protein expression technology that enables us to express the entire 21 subunits of Mediator. Second, we implemented the in vitro system in which the transcription process with Mediator can be recapitulated. Combining these two new methods with cryo-EM and single-particle analysis allows us to determine the mechanism of how Mediator orchestrates the assembly of transcription factors and Pol II onto promoter DNA for transcription initiation (Aim 1), to determine the structure of the 21-subunit Mediator in the context of transcription by cryo-EM and single-particle analysis (Aim 2), and to examine genetic interaction between Mediator and Pol II in vivo (Aim 3). Successful completion of the proposed project will reveal the fundamental mechanisms of Mediator in transcription regulation. The knowledge gained from yeast Mediator will be applied to that of higher eukaryotes including humans. Furthermore, our experimental strategy will be applied to that of human system in the future.