The long-term goal of this project is to understand the interactions between RNA polymerase II, its basal transcription factors, and the chromatin template that lead to accurate transcription initiation. The yeast Saccharomyces cerevisiae will be used as a model system for an integrated approach to the problem. Genetic approaches such as screens and selections for mutants, second-site suppressors, and synthetic lethality will be used to identify and verify in vivo interactions. Chromatin immunoprecipitation will analyze in vivo localization of factors. In vitro analysis of wild-type and mutant transcription factors will be performed using protein biochemical techniques (including affinity purification, enzymatic assays, reconstituted transcription, immobilized template assays, gel shift analysis, and footprinting). In the next period of the project, four major aims are proposed. (1) We will continue our analysis of the structure and function of basal factor TFIID (TATA-Binding Protein and its associated factors). Having developed an efficient TFIID purification, the role of several posttranslational modifications and TFIID-associated proteins will be studied. (2) A similar analysis will be carried out for TFIIH, allowing analysis of the interactions between its kinase and helicase activities. With efficient purifications of TFIID and TFIIH, we will continue our studies of yeast initiation complexes. (3) We will continue our studies of the TFIID-associated factor Bdf1. Having established that Bdf1 bromodomains interact with histone H4, we will study how this interaction affects transcription. We have recently discovered that Bdfl is phosphorylated by the kinase CK2 and that phosphorylation is required for Bdf1 function in vivo. The function of this modification will be studied in vivo and in vitro. (4) Yeast contains a RNA polymerase II-associated kinase known as Bur1 which resembles mammalian cdk9. However, our recent studies suggest that Bur1 may phosphorylate a substrate other than the CTD. Genetic data suggests that Bur1 is necessary to assist in transcription through chromatin and we will work to find the relevant Bur1 substrate(s) to determine its molecular function. Our work will help us understand the basic mechanisms of gene expression in all eukaryotic cells. Since improper gene expression is often at the root of cancer and developmental diseases, a clear understanding of the gene expression machinery will be required to develop effective therapeutics.