Genetic evidence for interdomain regulation of the phenol-responsive final sigma54-dependent activator DmpR.
The final sigma54-dependent DmpR activator regulates transcription of the dmp operon that encodes the enzymes for catabolism of (methyl)phenols. DmpR is expressed constitutively, but its transcriptional promoting activity is controlled positively in direct response to the presence of aromatic pathway substrates (effectors). DmpR has a distinct domain structure with the amino-terminal A-domain controlling the specificity of activation of the regulator by aromatic effectors (signal reception), a central C-domain mediating an ATPase activity essential for transcriptional activation, and a carboxyl-terminal D-domain involved in DNA binding. Deletion of the A-domain has been shown previously to result in an effector-independent transcriptional activator with constitutive ATPase activity. These results, in conjunction with the location of mutations within the A- and C-domains which exhibit an effector-independent (semiconstitutive) property, have led to a working model in which the A-domain serves to mask the ATPase and transcriptional promoting activity of the C-domain in the absence of effectors. To investigate the mechanism by which the A-domain exerts its repressive effect, we developed a genetic system to select positively for intramolecular second site revertants of DmpR. The results demonstrate (i) that mutations within the A-domain can suppress the semiconstitutive activity of C-domain located mutations and vice versa; (ii) that the C-domain located mutations do not influence the intrinsic ATPase and transcriptional promoting property of the C-domain in the absence of the A-domain; and (iii) that semiconstitutive mutations of the A- and C-domain have an additive effect. Taken together these results support a model in which the A-domain represses the function(s) of the C-domain by direct interactions between residues of the two domains.
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Department of Cell and Molecular Biology, Umeâ University, S-901 87 Umeâ, Sweden.
O'Neill
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Shingler
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D016384:Consensus Sequence; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D009876:Operon; D010452:Peptide Biosynthesis; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D010636:Phenols / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D051877:RNA Polymerase Sigma 54; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012808:Sigma Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C080133:DmpR protein, Pseudomonas; D009842:Oligopeptides; D010455:Peptides; D010636:Phenols; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C085706:FLAG peptide; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases