9304092 Weiss The soxRS (superoxide response) regulon of Escherichia coli an adaptation to oxidative stress. It includes the genes for a superoxide dismutase, endonuclease IV (a DNA repair enzyme), and glucose 6-phosphate dehydrogenase. Induction occurs in two stages. SoxR is first altered by an oxidative signal to become a transcriptional activator of SoxS. The overexpressed SoxS then activates the target genes of the regulon. The first question to be answered is whether or not the two-stage mechanism provides a temporally programmed response; i.e., are there genes other than soxS that are turned on directly by the activated SoxR and that are therefore expressed before those genes targeted by SoxS? To answer this question, 2-D gel protein patterns are being examined in regulon-constitutive (soxR) mutants that have a soxS deletion. The major part of this research deals with purification of SoxS and SoxR proteins from overproducing strains and a study of their interactions with their DNA binding sites and with RNA polymerase. Because the Sox S protein is a member of the large AraC family, so many analogs of which are being separately studied, investigations is limited to examining only its unique properties; thus, its specific recognition sequence will be delineated via DNA footprinting and mutational analysis of the for promoter region. SoxR, which is activated directly and/or indirectly by superoxide, will be studied more extensively. An attempt will be made to reconstruct SoxR-mediated soxS induction in vitro an to use it to identify the possible effector (s). The physical traits of the native SoxR protein will be compared with both mutationally activated and chemically activated forms to provide an insight into the mechanism of induction. The physical traits to be examined will include evidence of phosphorylation or proteolysis and changes in metal content, DNA footprint, titratable thiols, absorption spectrum, fluorescence, and multimer formation. %%% Because bacterial regulons are controlled in diverse ways, they provide many useful models for the coordinate regulation of dispersed genes in all organisms. Ultimate goals of the studies on the soxRS regulon are (a) the understanding of an additional system of multigene regulation and (b) the elaboration of the mechanism whereby organisms that grow in the presence of oxygen can deal with toxic by products of oxidative growth. ***
