The long-term objectives of the proposed project are to elucidate the mechanism of action and in vivo function of Mot1, an essential, conserved, transcriptional regulator in the yeast Saccharomyces cerevisiae. Mot1 forms a unique complex with the TATA-binding protein (TBP) and regulates TBP's function and DMA binding activity on a genome-wide scale. Mot1 is a member of a large family of evolutionary conserved nuclear ATPases (the Snf2/Swi2 family) involved in virtually all aspects of DMA metabolism. Defects in human Snf2/Swi2-related protein complexes contribute to certain cancers, Cockayne's Syndrome, a-thalassemia, and the most common form of X-linked mental retardation. Despite the ubiquitous occurrence of proteins in this family, their molecular mechanisms of action are not understood in detail, nor is it understood what roles many of these proteins play in vivo. Mot1 's ATPase activity is required to activate or repress transcription of specific genes in vivo. Consistent with its role as a repressor, Mot1 can dissociate TATA-binding protein (TBP)-DNA complexes in an ATP-dependent reaction. We propose that Mot1 can also activate transcription by using this activity to displace stably-bound, transcriptionally inactive forms of TBP from promoters. Such TBP recycling is proposed to provide quality control for transcription complex assembly and to ensure an adequate pool of free TBP for dynamic control of promoter activity genome-wide. Mot1- mediated activation also involves poorly understood cooperation with the NC2 and SAGA complexes. Biochemical, molecular biological, and genetic approaches will be used to test the recycling model and to define how Mot1, NC2 and SAGA cooperate in transcriptional control. Biochemical approaches will be used to test a specific and generally applicable model for Motl's catalytic mechanism in which ATP hydrolysis drives the interconversion of the ATPase between two different conformational forms. The proposed analysis of Mot1 function will lead to a better understanding of transcription complex dynamics and will provide general insight into how Snf2/Swi2-related proteins couple ATP hydrolysis to the generation of mechanical force.
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