The long term objectives of this application are to learn more about how chromatin structure controls transcription initiation in eukaryotic cells. Histones and certain classes of transcription factors that control chromatin structures have been demonstrated to play essential roles in normal transcription in all eukaryotes, ranging from yeast to humans. Mutations that impair the function of these factors have been associated with human diseases. The studies in this proposal, comprised of both genetic and biochemical approaches, focus on four sets of factors and how they function in vivo in the yeast, Saccharomyces cerevisiae. First, studies of the Snf/Swi complex will be conducted. Snf/Swi, a complex of twelve proteins, is believed to control transcription by remodeling nucleosomes, the fundamental unit of chromatin. The proposed studies will address the precise role of Snf/Swi at a promoter, the requirement for Snf/Swi for transcription of all S. cerevisiae genes, and the classes of mutants that impair their interaction with Snf/Swi. The mutants will be characterized genetically. The mutant histones will be purified and used to assemble mutant nucleosomes to be studied in vitro with respect to interaction with Snf/Swi and other factors. Third, studies of the Spt6 protein will be pursued. Spt6 is an essential protein, known to interact with histones. Spt6 and any associated proteins will be pursued. Spt6 is an essential protein, known to interact with histones. Spt6 and any associated proteins will be purified from S. cerevisiae cells. The associated proteins will be identified biochemically and studied genetically. To identify the role of particular motifs in Spt6, deletion mutations will be studied in vivo and in vitro. Finally, studies will be pursued of a putative histone acetyltransferase (HAT), Spt10. Spt10 is strongly required for the transcription of particular histone genes and its sequence suggests that it is a HAT. Site-directed mutations in the HAT consensus region of the SPT10 gene will be tested for function in vivo. HAT assays will be
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