Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.
The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. 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 its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.
O'Neill
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Department of Cell and Molecular Biology, Umeå University, Sweden.
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Shingler
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D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008024:Ligands; D010636:Phenols / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C080133:DmpR protein, Pseudomonas; D008024:Ligands; D010636:Phenols; D012808:Sigma Factor; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases