AROMATIC LIGAND-BINDING AND INTRAMOLECULAR SIGNALING OF THE PHENOL-RESPONSIVE SIGMA(54)-DEPENDENT REGULATOR DMPR

The Pseudomonas-derived sigma(54)-dependent regulator DmpR has an amin o-terminal A-domain controlling the specificity of activation by aroma tic effecters, a central C-domain mediating an ATPase activity essenti al for transcriptional activation and a carboxy-terminal D-domain invo lved in DNA binding. In the presence of aromatic effecters, the DmpR p rotein promotes transcription from the -24, -12 Po promoter controllin g the expression of specialized (methyl)phenol catabolic enzymes. Prev ious analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoti ng property until specific aromatic effecters are bound. Here, the aut onomous nature of the A-domain in exerting its biological functions ha s been dissected by expressing portions of DmpR as independent polypep tides. The A-domain of DmpR is shown to be both necessary and sufficie nt to bind phenol. Analysis of phenol binding suggests one binding sit e per monomer of DmpR, with a dissociation constant of 16 mu M. The A- domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by i ts normal derepression mechanism. The mechanistic implications of arom atic non-responsiveness of autonomously expressed A-domain, despite it s demonstrated ability to bind phenol, are discussed.