The long-term objectives of this project are to learn more about fundamental aspects of transcription initiation in eukaryotes. This process is strongly conserved in all eukaryotes, ranging from yeast to human. The studies will focus on protein factors discovered in the yeast, Saccharomyces cerevisiae, that control transcription initiation by RNA polymerase II. A combination of genetic and biochemical studies have shown that several transcription factors studied in the past, Spt proteins, are all present in a large protein complex. This complex, named SAGA, is critical for normal RNA polymerase II initiation in vivo. Experimental evidence strongly suggests that SAGA has more than one activity important for transcription. The proposed experiments are to use several approaches to analyze SAGA function in vivo. First, the role of SAGA at promoters will be tested by chromatin immunoprecipitation assays. These experiments will test models concerning the roles of different SAGA components in transcriptional activation. Second, the in vivo level of all mRNAs will be measured in wild-type strains and certain SAGA mutants to determine the genome-wide requirements for SAGA. These experiments will be done using DNA microarray technology. To test the possible overlap of SAGA with other characterized transcription factor complexes, the same approach will be used to analyze double mutants that affect more than one complex. Third, genetic approaches will be taken to identify new mutations that affect SAGA function. One of these mutant screens will allow testing deletions of every S. cerevisiae open reading frame. Finally, another function related to one SAGA component, Spt3, will be studied. This factor, Mot1, is not part of SAGA, but evidence suggests that Spt3 and Mot1 are both required for proper function of the general transcription factor, TATA-binding protein. These studies should reveal important aspects of transcriptional control in yeast. Given the strong conservation between yeast and humans, the results from these studies will be applicable to transcription in humans. These studies are relevant to human disease, since altered transcription in humans has been strongly implicated in diseases such as cancer.
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