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...

Since the growth of wild-type Escherichia coli in salicylate results in a multiple antibiotic resistance phenotype similar to that of constitutive mutants (Mar) of the chromosomal mar locus, the effect of salicylate on the expression of the marRAB operon was investigated. The amount of RNA hybridizing with a mar-specific DNA probe was 5 to 10 times higher in wild-type cells grown with sodium salicylate (5.0 mM) than in untreated controls. Untreated Mar mutants had three to five times more mar-specific RNA than wild-type cells did. When a Mar mutant was treated with salicylate, a 30- to 50-fold increase of mar-specific RNA was seen. In wild-type cells bearing a mar promoter-lacZ fusion on the chromosome, salicylate increased beta-galactosidase activity by sixfold. Thus, salicylate induces transcription of the marRAB operon. Other inducers of phenotypic multiple antibiotic resistance, e.g., benzoate, salicyl alcohol, and acetaminophen, but not acetate, also increased transcription from the mar promoter but to a lesser extent than did salicylate. Both in wild-type and mar-deficient strains, growth in salicylate resulted in increased antibiotic resistance, decreased permeation of the outer membrane to cephaloridine, increased micF transcription, and decreased amounts of OmpF. However, the magnitude of these changes was generally greater in wild-type (mar-containing) cells. Thus, salicylate and other compounds can induce transcription of the mar operon and, presumably, give rise to multiple antibiotic resistance via this pathway. However, salicylate can also activate an unidentified, mar-independent pathway(s) which engenders multiple antibiotic resistance.

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.