SoxS, an activator of superoxide stress genes in Escherichia coli. Purification and interaction with DNA.
A genetic response of Escherichia coli to nitric oxide or to superoxide-generating agents such as paraquat is controlled by the soxRS locus. The intracellular redox signals generated by these agents are sensed by the SoxR protein which, when activated, functions as a potent activator of soxS transcription. The resulting increased level of SoxS protein then activates approximately 10 genes that constitute the soxRS regulon. Although the SoxS protein is homologous to the COOH-terminal region of the AraC family of regulatory proteins, the mechanism by which SoxS protein activates the soxRS regulon promoters is unknown. We identified in extracts of cells expressing high levels of SoxS protein a DNA binding activity specific for fragments containing soxRS-regulated promoters. This binding activity was purified to physical homogeneity and proved to be the SoxS protein, as confirmed by NH2-terminal amino acid sequencing. The purified SoxS protein bound specifically to the promoters of the micF, zwf, nfo, and sodA genes. Multiple DNA-protein complexes were formed by SoxS in a concentration-dependent fashion with each of these promoters. This binding of SoxS protein also facilitated the subsequent binding of E. coli RNA polymerase to both the micF and the nfo promoters. The binding sites of SoxS in the zwf and micF promoters were identified by DNase I footprinting, which revealed an extended protected region immediately upstream of the respective -35 sites. These results indicate that the small SoxS protein (M(r) of only 12,900) is a direct transcriptional activator of the oxidative stress genes of the soxRS regulon, although the possible involvement of other proteins in transcription activation by SoxS has not been ruled out.
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
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D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000633:toxicity*; D010269:Paraquat / Q000633:toxicity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018085:Regulon; D013481:Superoxides / Q000633:toxicity*; D015534:Trans-Activators; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D013481:Superoxides; C070725:SoxS protein, E coli; D009569:Nitric Oxide; D003850:Deoxyribonuclease I; D010269:Paraquat