The overall objective of this work is to determine the molecular mechanisms that govern the regulation of transcription by RNA polymerase II. The general strategy is to focus on three Saccharomyces cerevisiae genes, SWI1, SWI2, and SWI3, which encode transcription factors whose activities are required for the function of a large number of gene- specific activators. Genetic studies indicate that one role for these SWI products may be to assist activators by antagonizing the inhibitory effects caused by nucleosomes and other chromatin components. Evidence is emerging that these SWI genes have been conserved during the evolution of multi-cellular eukaryotes and that these SWI products are required for the activity of several developmental regulators. The proposed studies may thus provide tools, both conceptual and concrete, for determining how many eukaryotic activators function. During my postdoctoral studies, significant progress has been made in my primary goal of determining the role that SWI products play in transcription. Specifically, it has been determined that SWI products play a global role in transcription by assisting the function of many activator proteins; that SWI products are required for synergistic activation of transcription by GAL4; and that SWI3 is associated with the glucocorticoid receptor in vitro. This proposal exploits the powerful genetic and biochemical opportunities available in yeast to test several hypotheses concerning the role of SWI products in transcription.
The first aim of this proposal is to determine which steps in the process of synergistic activation of transcription by GAL4 require SWI function.
This aim i s addressed by several in vivo methods, that include in vivo footprinting, the use of E. coli dam methylase as a probe of chromatin structure, and studies that use conditional alleles of SWI genes. The second objective is to test the hypothesis that SWI1, SWI2, and SWI3 function as components of a multisubunit complex by using coimmunoprecipitation assays, gel filtration chromatography, and genetics.
The third aim i s to test the hypothesis that SWI products associate with the GAL4 activator by using biochemical methods and a genetic screen. The fourth objective is to test the hypothesis that SWI1,2,3 functions as a novel helicase machine by determining the functional role of the SWI2 helicase sequence motifs both in vivo and in vitro. The results from the proposed studies will enrich our knowledge of the global role that SWI products play in transcription and provide valuable insights into the mechanisms for regulating transcription by RNA polymerase II.
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