The homologous MarA & SoxS transcriptional activators are synthesized by Escherichia coli and related bacteria in response to different environmental signals. In turn, they activate the transcription of an overlapping set of genes (the mar or sox regulons) which provides resistance to multiple antibiotics, superoxides and organic solvents. Control of the regulons is achieved by the presence of a binding site for the activators (the marbox) in the promoter regions of these genes. By detailed analysis of a number of regulon promoters, we (RG Martin & WK Gillette) have identified the minimal marbox as a 20bp sequence (consensus: AYnGCACnnWnnRYYAAAYn) and shown that it is functional in only one orientation at a given site relative to the promoter. The marbox is functional in the forward (F) orientation when it overlaps the -35 RNA polymerase (RNP) recognition hexamer and functions optimally when spaced 18 or 19 bp upstream of the -10 RNP recognition hexamer. The marbox is functional in the backward (B) orientation when spaced 38, 39 or 50 bp upstream of the -10 recognition hexamer. In one interesting case, the marbox is functional in the forward (F) orientation when spaced 30 bp upstream of the -10 hexamer. In every case, the marbox is on the same side of the DNA helix. Increasing or reducing the spacing by 2 or 3 bp, progressively reduces the transcriptional activation. Thus, the marbox also serves to align the activator on the DNA, presumably to permit interaction with RNP.In spite of their similarities, MarA & SoxS differ widely in the extents to which they activate particular promoters with the consequence that overexpression of SoxS leads to greater superoxide resistance than does overexpression of MarA. The marbox was found to be a critical element in this discrimination by assay of hybrid promoters containing the marbox from one gene and the core promoter of another. Furthermore, by sequential mutation of its marbox, a promoter that discriminated 35- fold in favor of SoxS was converted into one that did not discriminate. Band shift experiments showed that the extent of activation of a promoter is paralleled by the extent of MarA or SoxS binding to its marbox. Thus, differential recognition of closely related marbox sequences by the closely related activators is the primary basis for promoter discrimination. Discrimination may enable the cell to customize its response to the stresses that triggered synthesis of the activators. - Gene regulation; transcriptional activation; multiple antibiotic-resistance; salicylates; superoxide-resistance; Escherichia coli
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