The MarA protein and its homologs are transcriptional activators that mediate resistance to multiple antibiotics, superoxides and organic solvents in a number of gram-negative bacteria. Like the other members of the large archetypal AraC family of bacterial proteins, how they bind DNA and activate transcription is unknown. In collaboration with S. Rhee, D. Davies and R. G. Martin, the structure of MarA (from (Escherichia coli) bound to a cognate DNA oligonucleotide (marbox) has been solved by X-ray crystallography. MarA binds the DNA as a monomer by virtue of two helix-turn-helix (HTH) motifs. The HTH structures are each organized about a hydrophobic core. Numerous contacts between the recognition helices and the DNA major groove and overall shape complementarity between MarA and DNA provide for the somewhat loose sequence-specificity. By assaying the effects of alanine substitutions on MarA activity in vivo, W. Gillette has now demonstrated: (1) the importance of the hydrophobic cores; (2) differences in the roles of the two recognition helices; and (3) that single residues that contact bases may be replaced without loss of activity. This is consistent with our suggestion that multiple interactions contribute to the binding specificity. Our model provides an important framework for understanding DNA binding by other AraC proteins. Genes of the mar regulon are transcriptionally activated to different extents by MarA and its homolog, SoxS (~45% identity). Activation involves recognition by the activator of a highly degenerate marbox sequence that can be centered at various positions upstream of the -35 hexamer for RNA polymerase recognition. In contrast to dimeric transcriptional activators (e.g., CAP)which recognize symmetrical binding sites, we (R.G. Martin & W. Gillette) find that the marbox is asymmetrical: it is functional in one orientation when centered 51 or 42bp upstream of the transcriptional start site but functions in the other orientation when the marbox is 57bp or more upstream. We also find that the different extents of activation of a particular promoter by MarA or SoxS is dependent primarily on its ability to bind the marbox sequence and not on the downstream RNA polymerase recognition portions of the promoter. While MarA and SoxS generally are remarkably similar as activators, they do show strong differences at certain marboxes. Structure-function studies using MarA/SoxS chimeras are being pursued to localize the bases of these differences.
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