XylS-Pm promoter interactions through two helix-turn-helix motifs: identifying XylS residues important for DNA binding and activation.

The XylS protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. XylS belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain, made up of seven alpha-helices conforming two helix-turn-helix DNA-binding domains. alpha-Helix 3 and alpha-helix 6 are the recognition helices. In consonance with XylS structural organization, Pm exhibits a bipartite DNA-binding motif consisting of two boxes, called A and B, whose sequences are TGCA and GGNTA, respectively. This bipartite motif is repeated at the Pm promoter so that one of the XylS monomers binds to each of the repeats. An extensive series of genetic epistasis assays combining mutant Pm promoters and XylS single substitution mutant proteins revealed that alpha-helix 3 contacts A box nucleotides, whereas alpha-helix 6 residues contact B box nucleotides. In alpha-helix 3, Asn246 and Arg242 are involved in specific contacts with the TG dinucleotide at box A, whereas Arg296 and Glu299 contact the second G and T nucleotides at box B. On the basis of our results and of the three-dimensional model of the XylS C-terminal domain, we propose that Ser243, Glu249 and Lys250 in alpha-helix 3, and Asn299 and Arg302 in alpha-helix 6 contact the phosphate backbones. Alanine substitutions at the predicted phosphate backbone-contacting residues yielded mutants with low levels of activity, suggesting that XylS-Pm binding specificity not only involves specific amino acid-base interactions, but also relies on secondary DNA structure, which, although at another level, also comes into play. We propose a model in which a XylS dimer binds to the direct repeats in Pm in a head-to-tail conformation that allows the direct interaction of the XylS proximal subunit with the RNA polymerase sigma factor.