Summary Transcription activation, a key step in gene control, is the readout of many signaling pathways controlling cell growth, development and stress response and defects in activation cause many human diseases and syndromes. Activation typically results from factors containing activation domains (ADs) binding to coactivator complexes containing activator binding domains (ABDs). Understanding the nature of ADs, the specificity of AD-coactivator interactions, and mechanisms of coactivator cooperativity are necessary for understanding the molecular basis of gene regulation. The long-term goal of this project is to determine mechanisms used by gene-specific activators and coactivators to regulate RNA polymerase (Pol) II transcription. The objectives of this proposal are to determine: i) mechanisms of Mediator recruitment and its interaction with ADs, ii) mechanisms of cooperativity between Mediator and the coactivator SAGA, and iii) what constitutes a functional AD and determines the AD specificity for different coactivators and promoter types. This work will utilize an interdisciplinary combination of biochemical, structural, molecular, and computational approaches to examine transcriptional activation in S. cerevisiae. To understand these mechanisms, we will build upon several breakthrough concepts and methods developed in the past grant period. We will use a combination of genomics and biochemistry to identify a new mechanism of Mediator recruitment that is likely used at many genes. We will use orthogonal approaches to determine how the coactivator SAGA stimulates genome-wide transcription, either by direct interaction with the Pol II preinitiation complex (PIC) and/or by direct interaction with Mediator. We will determine the structural basis for AD-Mediator Tail domain interaction using a combination of biochemical and structural analysis. Finally, we will use a combination of biochemistry, genomics, and computational analysis to predict AD function and specificity and identify new mechanisms of AD-coactivator interaction. The expected outcome of these studies will be a molecular understanding of transcription activator specificity and function, mechanisms used by the coactivators Mediator and SAGA to promote transcription, and an understanding of how these factors function at different classes of genes –a leap forward in understanding widely conserved mechanisms of eukaryotic gene regulation.