A crystal structure for a member of the AraC prokaryotic transcriptional activator family, MarA, in complex with its cognate DNA-binding site is described. MarA consists of two similar subdomains, each containing a helixturn-helix DNA-binding motif. The two recognition helices of the motifs are inserted into adjacent major groove segments on the same face of the DNA but are separated by only 27 Å thereby bending the DNA by Ϸ35°. Extensive interactions between the recognition helices and the DNA major groove provide the sequence specificity.The AraC family of prokaryotic transcriptional regulators includes Ͼ30 proteins from different microorganisms, 18 from Escherichia coli alone (1). Members of this family control expression of a variety of genes by binding to specific promoter sites as either monomers or dimers. For AraC, the first transcriptional activator discovered (2), there are functionally independent DNA-binding and dimerization domains (3). Here we report the crystal structure of the MarA-DNA complex, providing the first structural basis for DNA-binding by the AraC family activator.MarA, a member of the AraC family, is a transcriptional activator of more than one dozen genes of the mar (multiple antibiotic resistance) regulon of E. coli (4-6). It consists of 129 amino acids, exists as a monomer in solution, does not contain a dimerization domain, and binds to an asymmetric, degenerate 20-bp DNA sequence (5, 7). This contrasts with other prokaryotic transcriptional regulators that generally act as dimers and bind tightly to unique direct or inverted repeat sequences (8-10). METHODSSelenomethionyl MarA with an N-terminal polyhistidine tag was expressed in an E. coli met Ϫ auxotropic strain, B834(DE3) (Novagen), and was purified as described for the native MarA (4). The purified MarA (Ϸ30 mg͞liter cell culture) was dissolved in a solution of 50% (vol͞vol) glycerol, 50 mM Hepes (pH 8.0), and 0.5 M NaCl and was stored at Ϫ20°C for further use. The synthetic oligonucleotides (purchased from Keck Oligonucleotide Synthesis Facility of Yale University) were purified by reverse-phase HPLC (C4 column) by using a linear gradient of acetonitrile in 0.1 M triethylammonium acetate (pH 7.0). The MarA-DNA complex was prepared by first mixing equal molar amounts of the two complementary oligonucleotide strands at room temperature and then adding MarA to a solution of the duplex DNA at a 1:1.2 M ratio. Solubility of MarA was greatly enhanced by forming the complex with DNA. The MarA-DNA complex was dialyzed at 4°C against a buffer of 10 mM Hepes (pH 8.0) and 10 mM NaCl and was concentrated to 10 mg of MarA͞ml of solution.The crystals were grown at room temperature by the hanging drop method using the sparse matrix screen (11) from Hampton Research (Riverside, CA). The best diffracting crystals of a size 0.3 ϫ 0.2 ϫ 0.1 mm were obtained with a 22-bp double stranded DNA fragment (see Fig. 1B) by micro-seeding crystals in the presence of the mother liquor, 12% PEG 8000, 100 mM sodium cacodylate (pH 6.5), and 100 mM c...

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SummaryThe promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely de®ned because of its degeneracy and asymmetry and seemingly variable location with respect to the À10 and À35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more de®ned marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the À35 hexamer are oriented opposite those closer to the À35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.

Elevated expression of the marORAB multiple antibiotic-resistance operon enhances the resistance of Escherichia coli to various medically significant antibiotics. Transcription of the operon is repressed in vivo by the marR-encoded protein, MarR, and derepressed by salicylate and certain antibiotics. The possibility that repression results from MarR interacting with the marO operator-promoter region was studied in vitro using purified MarR and a DNA fragment containing marO. MarR formed at least two complexes with marO DNA, bound >30-fold more tightly to it than to salmon sperm DNA, and protected two separate 21-bp sites within marO from digestion by DNase I. and weakened the interaction of MarR with sites I and II. Thus, repression of the mar operon, which curbs the antibiotic resistance of E. coli, correlates with the formation of MarRsite I complexes. Salicylate appears to induce the mar operon by binding to MarR and inhibiting complex formation, whereas tetracycline and chloramphenicol, which neither bind MarR nor inhibit complex formation, must induce by an indirect mechanism.

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Transcriptional activation of the promoters of the mar/soxRS regulons by the sequence-related but independently inducible MarA and SoxS proteins renders Escherichia coli resistant to a broad spectrum of antibiotics and superoxide generators. Here, the effects of MarA and SoxS on transcription of the marRAB promoter itself were assayed in vitro by using a minimal transcription system and in vivo by assaying ␤-galactosidase synthesized from marR::lacZ fusions. Purified MarA and MalE-SoxS proteins stimulated mar transcription about 6-and 15-fold, respectively, when the RNA polymerase/DNA ratio was 1. Purified MarA bound as a monomer to a 16-bp ''marbox'' located 69 to 54 nucleotides upstream of a putative RNA initiation site. Deletion of the marbox reduced MarA-mar binding 100-fold, abolished the stimulatory effects of MarA and SoxS on transcription in vitro, and reduced marR::lacZ synthesis about 4-fold in vivo. Deletion of upstream DNA adjoining the marbox reduced MarA binding efficiency 30-fold and transcriptional activation 2-to 3-fold, providing evidence for an accessory marbox. Although MarA and the mar operon repressor, MarR, bound to independent sites, they competed for promoter DNA in band shift experiments. Assays of marR::lacZ transcriptional fusions in marRAB deletion or soxRS deletion strains showed that the superoxide generator paraquat stimulates mar transcription via soxRS and that salicylate stimulates mar transcription both by antagonizing MarR and by a MarR-independent mechanism. Thus, transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS.

Summary Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.

Expression of the marA or soxS genes is induced by exposure of Escherichia coli to salicylate or superoxides, respectively. This, in turn, enhances the expression of a common set of promoters (the mar/soxRS regulons), resulting in both multiple antibiotic and superoxide resistance. Since MarA protein is highly homologous to SoxS, and since a MalE-SoxS fusion protein has recently been shown to activate soxRS regulon transcription, the ability of MarA to activate transcription of these genes was tested. MarA was overexpressed as a histidinetagged fusion protein, purified, cleaved with thrombin (leaving one N-terminal histidine residue), and renatured. Like MalE-SoxS, MarA (i) activated the transcription of zwf, fpr, fumC, micF, nfo, and sodA; (ii) required a 21-bp ''soxbox'' sequence to activate zwf transcription; and (iii) was ''ambidextrous,'' i.e., required the C-terminal domain of the ␣ subunit of RNA polymerase for activation of zwf but not fumC or micF. Thus, the mar and soxRS systems use activators with very similar specificities and mechanisms of action to respond to different environmental signals.