All known organisms require divalent zinc [Zn(ll)] and yet this essential nutrient can be toxic. Recent epidemiological studies suggest that human Zn(ll) deficiency is a worldwide health problem that may be associated with an increased risk of cancer. Human Zn(ll) deficiencies may also be linked to immunological, reproductive and genotoxic effects. Organisms ranging from bacteria to mammals maintain intracellular Zn(ll) levels within a functional range through homeostatic mechanisms that are regulated at the level of gene expression. In humans and other higher eukaryotic organisms, the metal-response element-binding transcription factor-1 (MTF-1) coordinates the expression of genes involved in Zn(ll) homeostasis, metal ion detoxification and protection against oxidative stress. The overall goal of these proposed studies is to elucidate the molecular details of Zn(ll)-induced transcriptional activation of MT gene expression by MTF-1. Although the reversible activation of MTF-1 transcription by Zn(ll) excess is at least partially regulated at the level of coordination to metal sites within the Cys2His2 zinc finger DMA binding domain, recent studies suggest other co-factors and regions of the protein may be involved in MTF-1 metalloregulatiory function. However, the relative contributions and molecular details of the regulatory mechanisms involving each of these regions are not well understood. Herein, we are elucidating the molecular mechanisms of Zn(ll)- responsive activation of MT gene expression by MTF-1 potentially involving DNA binding, nuclear localization, transcriptional activation, and nuclear export domains within the protein. High-resolution NMR- based structural methods are complimenting biophysical, biochemical, and molecular biology approaches to address the following Specific Aims: 1) Assess the structural determinants of Zn(ll) binding to the MTF-1 zinc fingers and elucidate the role of Zn(ll)-protein binding in MTF-1 nuclear localization.;2) determine the solution structure and binding affinity of the MTF-1 zinc finger domain-MRE DMA complex, and assess the metalloregulatory contribution of finger linker peptides in DNA binding, and 3) further define the Zn(ll)- specific p300 binding site in MTF-1, explore the structural basis for this interaction, and assess the relationship of MTF-1 -p300 interaction to Zn(ll) homeostasis.