Probing the Escherichia coli transcriptional activator MarA using alanine-scanning mutagenesis: residues important for DNA binding and activation.

The MarA transcriptional activator binds to a 20 bp asymmetric degenerate sequence (marbox) located at different positions and orientations within the promoters of the genes of the Escherichia coli mar regulon. Solution of the MarA-marbox X-ray crystallographic structure suggested the presence of base-specific and non-specific interactions between the marbox and two helix-turn-helix (HTH) motifs on the monomeric MarA. Here, we use alanine-scanning mutagenesis and DNA retardation analysis to: (i) evaluate the contacts between MarA and the marboxes of five differently configured mar regulon promoters; (ii) assess the role of conserved hydrophobic amino acid residues for MarA activity; and (iii) identify residues required for RNA polymerase activation. These analyses revealed that the phosphate-backbone contacts and hydrogen bonds with the bases of the marbox are more significant for DNA binding than are the van der Waals interactions. While both N and C-terminal HTH motifs make essential contributions to binding site affinity, MarA is more sensitive to alterations in the N-terminal HTH. In a similar way, the activity of MarA is more sensitive to alterations in the hydrophobic core of this HTH. Solvent-exposed amino acid residues located at many positions on the MarA surface are important for activity. Some of these residues affect activity on all promoters and thus, are implicated in maintaining MarA structure whereas several solvent-exposed amino acids not involved in DNA binding were important for MarA activity on specific promoters. The pattern of activation defects defined a class II promoter-specific activating region. However, a localized class I activating region was not apparent. These results suggest that MarA activates transcription by at least two distinct mechanisms. Furthermore, the important role of phosphate contacts in marbox affinity suggests that indirect readout contributes to binding site recognition by MarA.