Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling

Pseudomonas derived sigma(54)-dependent regulators DmpR and XyIR control th e expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effecters controls the acti vities of these transcriptional activators. Previous work has derived a com mon mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repress ion of an intrinsic ATPase activity essential for its transcription-promoti ng property and allows productive interaction with the transcriptional appa ratus. Here we dissect the A-domains of DmpR and XyIR by DNA shuffling to i dentify the region(s) that mediates the differences in the effector specifi city profiles. Analysis of in vivo transcription in response to multiple ar omatic effecters and the in vitro phenol-binding abilities of regulator der ivatives with hybrid DmpR/XyIR A-domains reveals that residues 110 to 186 a re key determinants that distinguish the effector profiles of DmpR and XyIR . Moreover, the properties of some mosaic DmpR/XyIR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from t he ability to promote transcription. Hence, novel aromatic binding properti es will only be translated into functional transcriptional activation if ef fector binding also triggers release of interdomain repression.