A research program concerned with the mechanism of metal ion regulated gene expression and metalloprotein biosynthesis/assembly is proposed here. The project focusses on two proteins, plastocyanin and cytochrome c6 that are photosynthetic electron transfer catalysts. We have shown that the biosynthesis of these proteins is subject to a metal-sensing regulatory system where Cu, at nanomolar concentrations, is the signal which elicits as a response the accumulation of one protein (the Cu-containing plastocyanin) and the repression of the other (functionally equivalent heme protein cyt c6). The detailed characterization of the molecular constituents and mode of operation of this Cu-dependent regulatory system is the objective of our work. We will identify copper-regulatory elements (CREs) in the Chlamydomonas gene for cyt c6 by in vivo deletion analysis and identify sequence-specific DNA binding proteins (BPs) that function as Cu-dependent transcriptional regulators in vitro. The proposed role of plastocyanin as a """"""""Cu-sink"""""""" will be verified by the study of strains containing decreased or increased levels of holoplastocyanin. To gain a more complete and comprehensive picture of regulatory circuits, we will isolate and characterize mutants that display altered regulation of cyt c6, and we will examine the regulatory features of two other Cu-responsive proteins -- the reciprocally regulated plastocyanin of Scenedesmus, and a coordinately regulated 30 kDa protein of Chlamydomonas. The post- translational pathway for maturation of plastocyanin and cyt c6 will be investigated by molecular and genetic dissection. And, we will initiate a structural comparison of plastocyanin and cyt c6 -- structurally distinct but functionally equivalent electron transfer catalysts. Transition metals are essential components of biological systems. By elucidating their utilization and function (as catalysts and regulators), our work contributes to the understanding of fundamental biochemical processes and has practical applications with respect to metal-metabolism based diseases and the potential creation/modification of metal- resistance/tolerance. My long-term goal is to establish myself as an independent investigator in the area of metalloprotein biosynthesis, structure and function. In the next five years, I will pursue the above projects and enhance the research capabilities of may laboratory by incorporating genetic and structural studies into our program. In combination with established molecular and biochemical techniques, these new approaches will expand our experimental base and lead to new avenues of investigation. Although my intellectual background in genetics and structure is strong, I need to acquire practical experience and strengthen my research expertise in these relevant disciplines. An RCDA, in allowing for my intensive research involvement, and providing time for developing experience and skills in new areas would significantly promote the development of my career. The department of Chemistry and Biochemistry has provided substantial space, funds and facilities for my research program. The department fully supports the application for an RCDA in order to promote my research career and will release me from all undergraduate teaching assignments and from graduate (non-research related) """"""""core"""""""" teaching responsibilities.
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