Oxidative stress is widespread in biology and associated with aging and carcinogenesis. In response to such stress, cells modulate gene expression to escape damage or repair oxidative injury. Gene expression linked to oxidative stress has been described but few molecular signaling mechanisms are defined. The investigators have shown that E. coli SoxR protein is a novel transcription activator triggered by oxidation, or by nitrosylation of its essential iron-sulfur centers. Oxidation or nitrosylation of the SoxR [2Fe-2S] centers transduces signals of cellular exposure to pro-oxidants or nitric oxide (NO) into expression of antioxidant, DNA repair, and other defense genes. In this proposal the investigators wish to define the biological, biochemical, and structural basis for this redox/NO signal transduction and response. They will explore biochemically and genetically the cellular systems that switch off activated SoxR by reduction, or processing of the nitrosylated form. The reactivity and control of SoxR will be related to biochemical and physical analysis of SoxR protein structure and the isolation of novel mutant forms of the protein and of stable fragments. The detailed interaction of SoxR with DNA will be explored by in vitro methods, and new approaches will be developed to test whether DNA supercoiling can drive transcription activation by SoxR in vivo as they have recently shown in vitro. They will use a novel nanomechanical device to test explicitly and directly the hypothesis that activated SoxR triggers transcription by localized DNA untwisting. They will investigate in vitro whether transcription dislodges SoxR from DNA to allow redox equilibration with the protein in solution. They will test whether NO-related compounds such as nitrosothiols, can also activate SoxR by nitrosylating its iron-sulfur centers. The role of SoxRS-inducible genes that mediate cellular resistance to NO will be identified, as well as novel resistance genes controlled by SoxS and the homologous Rob protein. Using the Rob structure they determined recently, they will test possible activating ligands for this proposed environmental response protein. The knowledge gained from these studies will illuminate approaches to dissecting the mechanisms and roles of redox and NO signal transduction in all organisms.
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