The goal of this research is to elucidate the mechanisms of eucaryotic gene regulation. The system chosen to study is the multi-protein CCR4-NOT complex, which affects gene expression both positively and negatively. It consists of two complexes, 1.9 and 1.0 mD in size, that are distinct from other transcriptionally important groups of proteins, such as the SNF/SWI and SAGA complexes, TFIID, and the RNA polymerase II holoenzyme. The smaller of the CCR4-NOT complexes contains CCR4, CAF1, the five NOT proteins and several unidentified proteins. The repressive effects of the CCR4-NOT proteins appear to occur through destabilizing TBP association with non-canonical TATAA sequences, probably through their direct interaction with TFIIA, TAFs or other TBP-associated factors. The SRB9-11 holoenzyme subcomplex also displays physical and functional interactions with the CCR4-NOT complex. The central focus of this grant proposal is three-fold: 1) What are the components and physiological roles of the two CCR4-NOT complexes? 2) How do the CCR4-NOT proteins affect the function of TBP and its associated factors? and 3) How is the CCR4-NOT complex required for activated transcription? The 1.9 mD CCR4-NOT complex will be isolated, its components cloned following mass spectrometric analysis, and their functions characterized. The mechanism by which the CCR4-NOT complex affects the action of TBP and its associated factors will be elaborated by several biochemical studies. Genetic screens will be undertaken to identify new factors or novel interactions between the CCR4-NOT complex and transcriptionally important proteins. CCR4 activator function will be explored through characterizing its unique C- and N-terminal interactions with SRB9-11, SPT5, TAF60, and other proteins. The key features of the ADH2 promoter which are responsive to the CCR4-NOT complex will be identified to determine how the CCR4-NOT complex controls the ADH2 gene in a positive manner.
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