The long-term goal of this proposal is to elucidate the dynamic interactions that regulate early events in eukaryotic transcription initiation by RNA polymerase II. Assembly of the TFIIA-TFTID complex is an early step in preinitiation complex formation that can be modulated by interactions with numerous activators, coactivators, repressors, and corepressors. In this application I will emphasize the importance of the general transcription factor TFIIA as a promoter-selectivity factor, a modulator of TBP-interactions, and as a target of post-translational regulation. Evidence suggests that TFIIA has promoter-specific functions that are required for many, but not all RNA polymerase II transcription in vivo. The promoter sequences and cellular conditions that specify a requirement for one or another TFIIA function are not known. We propose to characterize promoter-specific functions of TFIIA using genomic array analysis of mechanistically defined TFIIA mutant alleles. TFIIA mutants have been characterized with defects in TBP-binding, TAFinteractions, NC2 derepression, DNA interactions, and phosphorylation acceptor sites. We will test these functionally different mutant alleles of TFIIA for genome-wide defects to identify specific promoters dependent upon these transcription activities. We will investigate the mechanistic basis for TFIIA promoter-specific functions using both in vivo and in vitro promoter-association assays. The role of TFIIA in regulating the recruitment of general transcription factors, TFIID TAFs, SAGA, the Mediator/SRB complex, and TBP-specific repressors will be investigated. We have also found that TFIIA phosphorylation can stimulate interactions with TBP and facilitate high level transcription in vivo. TFIID has been shown to be a candidate TFIIA kinase, and we propose to explore the regulatory function of TFIIA phosphorylation in the transcription cycle of several genes. Post-translational regulation of promoter core binding factors, like TFIIA and TBP, have not been investigated in significant detail. Although this grant focuses on the very specific questions regarding the role of TFIIA in core promoter recognition, we consider TFIIA to be central to many of the larger questions regarding the mechanisms of eukaryotic transcription activation and repression. Using genetic and biochemical approaches, this research proposal aims to elucidate novel mechanisms of transcription regulation and understand the complex interplay between promoter core binding factors and the coactivators and corepressors that regulate cell growth and differentiation.
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