This study will concentrate on the effects of oxygen-derived free radicals. These oxidants are generated not only by lonizing radiation but also by normal aerobic metabolism in almost all cells. They have been implicated as causes of mutations, cancer, aging and of tissue damage associated with inflammation. This proposal will focus on a specific multigene system in Escherichia coli that is induced by compounds that generate superoxide. This global response includes the genes for the DNA repair enzyme endonuclease IV, for a superoxide dismutase, and for glucose-6- phosphate dehydrogenase, all of which are positively regulated at a transcriptional level by soxR and soxS, newly discovered genes that have been cloned, mapped and sequenced in this laboratory. The soxRS region will be transcriptionally mapped and tested for exogenous and endogenous regulation. Existing mutations will be sequenced and additional specific ones produced if needed. The proteins will be purified and their effect will tested in vitro on the transcription of one of the target genes, that for endonuclease IV (nfo). In the likely event that one or both or the proteins bind on the nfo promoter region, then the contact points will be identified via footprinting, in vitro modification of the DNA, sequencing of promoter-constitutive mutations, and a survey of target sites in other genes. Physical studies of the purified proteins will center around possible proteins-proteins and protein-nucleic acid interactions, conformational changes accompanying activation, and the function of four close cysteines in soxR. Posttranscriptional control will be explored by connecting the nfo (endonuclease IV) target gene to foreign promoters and possible feedback loops will be examined by measuring nfo transcription and translation in cells with different copy numbers of the gene. If the entire system cannot be reconstructed in vitro, then additional genes in this pathway will be sought through mutation, employing gene fusions as indicators. //
