The experiments in this proposal will further our understanding of several important aspects of transcriptional activators and signal transduction. An understanding of transcriptional activators and the pathways that regulate their activities are important in health considerations, most prominently because their derangement can lead to cancer and probably other diseases. In fact, several oncogenes are transcriptional activators. The proposed experiments continue to aim toward a detailed understanding of two model cases that we have developed in the yeast system. These cases have direct relevance to activators in higher cells, including mammals. The proposal will focus on structural and genetic studies of the DNA- binding domain of the yeast transcriptional activator, HAP1, and molecular and biochemical analysis of the heteromeric complex, HAP2/314. The pathway by which heme activates HAP1 will be analyzed. The binding of heme to a short response element will be studied structurally. Also, genes encoding cellular repressors that participate in the process will be cloned. Issues relating to DNA-binding of HAP1 will be addressed, including a structural analysis of HAP1 bound at two different sequence elements, and an investigation of why the protein prefers to bind asymmetrically to directly repeated DNA half sites. Targets of the HAP1 DNA-binding domain that are important in transcriptional activation will be identified. A novel component of the HAP2/3/4 heteromeric complex, which binds to the CCAAT box, will be cloned. The complex will then be dissected further to determine what surfaces are involved in protein-protein interaction, and why three subunits are required for CCAAT-binding in this novel DNA- binding domain.
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