The goal of this work is to understand, in precise molecular terms, the mechanism of action of a yeast transcriptional repreessor, the Alpha 2 protein. This protein turns off transcription of a group of yeast cell-type specific genes called the a-specific genes. The Alpha 2 protein binds tightly and specifically to an operator located upstream of an a-specific gene, STE6. This operator brings about repression when located within or entirely upstream of a """"""""test"""""""" promoter. Specific models developed to explain this repression at a distance will be rigorously tested by the proposed experiments. Specifically, the distance (relative to the test promoter) over which the operator can function will be determined. Constraints (if any) on the promoter/operator geometry required for repression will be defined. Mutant operators will be generated to determine if the operator is simply an alpha 2 recognition sequence or, as suspected, contains some additional feature required for repression. Milligram quantities of Alpha 2 will be purified to homogeneity and used to determine whether or not Alpha 2, when it binds to its operator, unwinds the DNA and/or bends the DNA. Molecular probe experiments will reveal structural feature of the Alpha 2-operator interaction. The purified Alpha 2 protein will be used as an affinity reagent in an attempt to identify, purify and characterize other components involved in the repression mechanism. The physiological role of these other components will be deduced from genetic experiments. The mechanism by which Alpha 2 acts in combination with a second regulatory protein, a1, will be determined. Together, these proteins turn off transcription of a group of genes known as the haploid-specific genes. We will purify the Alpha 2/a1 regulatory activity, determine its composition and study its properties. Finally, a yeast transcription system will be reconstituted in vitro in order to determine the precise step or steps in transcription blocked by the action of Alpha 2. The proposed studies of Alpha 2 will provide a detailed molecular picture showing how cell specialization is maintained in the yeast Saccharomyces cerevisiae. A basic understanding of the molecular events underlying cell specialization is essential for understanding how the process can fail giving rise to pathological conditions.
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