The goal of the proposed research is to obtain a complete understanding at the molecular level of a complex regulatory circuit which controls nitrogen metabolism in Neurospora crassa. The multiple functions of the global-acting regulatory protein NIT2, which shares homology with human transcription factors GATA-1, -2, -3, and -4 will be examined in detail. The function and regulation of trans- acting nitrogen regulatory proteins, NIT2, NIT4, and NMR will be elucidated by site-directed and conventional mutagenesis, followed by analyses both in vitro and in vivo. A major focus is upon multiple functional protein-protein interactions in which the global-acting NIT2 participates, between the NIT2 and NMR regulatory proteins and between the NIT2 and NIT4 proteins. These interactions will be investigated by direct biochemical studies , by mutational analysis, and by use of the yeast two-hybrid system. The exact requirements for DNA binding by the pathway-specific NIT4 protein will be determined, and the nature of the NIT4 dimerization domain will be examined. NIT2 and/or NIT4 binding elements will be inserted into the promoter of a new test gene to assess their individual and joint function. Factors responsible for mediating glutamine-dependent nitrogen catabolite repression will be identified by genetic and biochemical studies, and a potential post-translational modification of NIT2 or NMR will be examined. A combined molecular and genetic analysis will be used to understand the molecular basis for the autogenous control of gene expression exerted by the enzyme nitrate reductase, which may involve interaction in a complex feedback loop with the transcription factor NIT4. The function of two new GATA factors will be determined.
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