Novel effector control through modulation of a preexisting binding site ofthe aromatic-responsive sigma(54)-dependent regulator DmpR

The Pseudomonas derived sigma(54)-dependent DmpR activator regulates transc ription of the (methyl)phenol catabolic dmp-operon, DmpR is constitutively expressed, but its transcriptional promoting activity is positively control led in direct response to the presence of multiple aromatic effecters. Prev ious work has led to a model in which effector binding by the amino-termina l region of the protein relieves repression of an intrinsic ATPase activity essential for its transcriptional promoting property. Here, we address whe ther the observed differences in the potencies of the multiple effecters (i ) reside at the level of different aromatic binding sites, or (ii) are medi ated through differential binding affinities; furthermore, we address wheth er binding of distinct aromatic effecters has different functional conseque nces for DmpR activity. These questions were addressed by comparing wild ty pe and an effector specificity mutant of DmpR with respect to effector bind ing characteristics and the ability of aromatics to elicit ATPase activity and transcription, The results demonstrate that six test aromatics all shar e a common binding site on DmpR and that binding affinities determine the c oncentration at which DmpR responds to the presence of the effector, but no t the magnitude of the responses. Interestingly, this analysis reveals that the novel abilities of the effector specificity mutant are not primarily d ue to acquisition of new binding abilities, but rather, they reside in bein g able to productively couple ATPase activity to transcriptional activation . The mechanistic implications of these findings in terms of aromatic contr ol of DmpR activity are discussed.