The long-range objective of this proposal is to understand the process of transcription initiation by RNA polymerase II (Pol II) in vivo. The focus is on the function of TATA-box binding protein (TBP), a highly conserved protein found in all eukaryotes. Binding of TBP to the TATA-box in promoters of genes transcribed by Pol II initiates assembly of the remainder of the transcription machinery. Due to this critical function, it is important to identify factors that interact with TBP and determine how these interactions regulate TBP activity. A combination of techniques, including classical and molecular yeast genetics, recombinant DNA technology, and protein biochemistry, will be used to accomplish the following. First, TBP functions involved in activated transcription by Pol II in vivo will be determined. Two distinct mechanistic classes of activation-defective TBP alleles have been described: defective for recruitment of TBP to the TATA-box; and defective after TBP arrives at the TATA-box. Interactions between these TBP mutants and key components of the transcription machinery will be examined. Also, unlinked suppressors of these mutations will be isolated, the genes cloned and studied. Second, the role of TFIIA in transcription will be investigated. Since the TBP-TFIIA interaction is essential for activation in vivo, factors capable of recognizing TFIIA or the TFIIA-TBP complex, will be identified and characterized. Additional functions of TFIIA will be defined by mutational analysis and subsequent suppression of the mutant phenotypes. Third, the mechanisms of transcription at TATA-lacking Pol II promoters will be defined in vivo. Not all Pol II promoters contain TATA-boxes, although TBP is still required. Using a TBP mutant specifically defective for TATA-less transcription, factors will be identified (by genetic and biochemical approaches), that play a unique role in transcription from TATA-less promoters. Fourth, genetic selections will be used to identify new alleles of TBP defective in other aspects of Pol II transcription, thereby uncovering novel TBP functions. In summary, the proposed experiments are intended to provide information on the function of TBP and TBP-interacting factors in eukaryotic gene regulation. The factors and mechanisms of transcription initiation are conserved from yeast to humans. Thus, these results will be directly relevant to higher eukaryotes, where changes in gene expression are critical aspects of cell growth, development, and the response to environmental stimuli.
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