The development of therapeutics to intercede in the mis-expression of genes that debilitate our health will be aided by a thorough understanding of the molecular events controlling gene expression. One such step is the dissociation of TATA binding protein (TBP) dimers. TBP is essential for the expression of all eukaryotic genes. Dimerization of TBP (and its multi-subunit counterpart, TFIID) prevents TBP from accessing promoter DNA, and thus prevents transcription complex assembly and ultimately expression of the gene. Mutations in the dimer interface (TBP/EB mutants) increase the expression of induced genes but not induced genes, which is consistent with the notion that activators accelerate this rate- limiting step in gene expression. Preliminary data suggest the existence of numerous regulators of TBP dimerization. The long term objective of this research is to understand the interplay among cellular transcriptional regulators, TBP, and TBP-associated factors that leads to the positive and negative regulation of TBP dimers. Such research will reveal how TBP dimerization and its regulation is integrated into the multiple layers of regulation imposed on every gene. The following specific aims are directed at understanding to what extent TBP dimerization prevents unregulated gene expression in yeast, and the mechanisms by which dimerization is regulated: 1) Identify TBP/EB- responsive genes. 2) Define the mechanism by which Brf1 dissociates TBP dimers. 3) Purify, characterize, and clone TDI, a regulator of TBP dimers. 4) Define how TFIIA regulates TBP and TFIID dimers. To identify TBP/EB responsive genes, S1 analysis and microarray technology will be used to measure the expression level of yeast genes harboring the TBP/EB mutants. Similar analyses will be performed in strains defective for particular activators and repressors, so as to uncover additional genes where dimerization strains defective for particular activators and repressors, so as to uncover additional genes where dimerization may be involved. The role of Brf1 and TFIIA in disrupting TBP dimers will be examined through mutational analysis and use of pulldown and crosslinking dimerization assays. Dimer mutants will be tested for suppression of Brf1 and TFIIA mutants in yeast. Standard and affinity chromatography will be used to purify TDI from HeLa cells. Its gene(s) will be cloned by standard molecular techniques, and structure/function analyses performed on the recombinant protein.
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