Oxidative stress is widespread in biology and related to aging and degenerative disease. Cells modulate gene expression in the face of such stress to escape or repair oxidative injury. Molecular signal transducing mechanisms that link oxidative stress to transcriptional control have not yet been defined. This issue will addressed for the redox-responsive SoxRS system that triggers a cascade of gene expression in E. coli subjected to superoxide or nitric oxide stress. SoxR protein, which contains an iron-sulfur cluster essential for transcriptional activation, will be analyzed by visible spectroscopy and electron paramagnetic resonance. Reactions of specific free radicals (superoxide and nitric oxide) with SoxR will be examined. This information will be coupled to that from in vitro studies of the SoxR interaction with DNA and transcriptional activation, which we hypothesize occurs via bending and twisting of the promoter DNA structure. The signaling mechanism that links a redox-active FeS center in a transcription factor to the stimulation of RNA polymerase will be defined by testing explicitly whether SoxR activity is controlled by the oxidation state or stability of its FeS center. Genetic approaches will be employed to explore the roles of individual amino acid residues and polypeptide regions in this gene activation process. Cellular activities will be identified that may maintain SoxR in the non- activated state awaiting a physiological induction signal. The transcription activating mechanism of the SoxR-induced SoxS protein, which is directly responsible for switching on the 10-12 promoters of this regulon, will be investigated. The principal investigator will define further the role of SoxR/SoxS activation in bacterial resistance to nitric oxide generated during immunological attack and determine whether such a regulatory system is widely distributed in pathogenic strains. Together, these studies will provide an integrated understanding of cellular mechanisms that control expression of oxidative stress genes and of their biological roles.
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