Cooperative binding of DctD to the dctA upstream activation sequence of Rhizobium meliloti is enhanced in a constitutively active truncated mutant.
DctD, a sigma54-dependent, two-component regulator, binds to promoter distal (A) and promoter proximal (B) sites in an activation sequence located upstream of the dctA promoter. We report gel filtration and quantitative DNase I footprint experiments supporting a model in which DctD2 binds to these sites cooperatively. The global analysis of upstream activation sequences containing sites A and B, A and B one-half helical turn out of phase, and only B yielded values for the intrinsic and cooperative binding free energies of DeltaG0A = -9.5 +/- 0.3, DeltaG0B = -11.2 +/- 0.2, and DeltaG0AB = -2.5 +/- 0.5. A separate analysis of data from upstream activation sequences containing site A and a point mutant of site B, and site A and mutant site B one-half helical turn out of phase confirmed the estimate of cooperativity, yielding free energy values of DeltaG0A = -9.4 +/- 0.2, DeltaG0B(G-->C) = -10.0 +/- 0.2, and DeltaG0AB(G-->C) = -2.2 +/- 0.4. We previously showed that removing the two-component receiver domain from DctD, making DctDDelta(1-142), yields a constitutively active truncated protein. Global analysis of binding data for DctDDelta(1-142) showed that this constitutively active mutant has intrinsic binding energies equal to that of the inactive DctD protein, but that it displays significantly higher cooperativity (DeltaG0A = -9.4 +/- 0.6, DeltaG0B = -11.1 +/- 0.3, and DeltaG0AB = -3.8 +/- 0.6.).
Scholl
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Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Nixon
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BT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D029382:Dicarboxylic Acid Transporters; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029382:Dicarboxylic Acid Transporters; D014157:Transcription Factors; C073786:dctA protein, Sinorhizobium meliloti; C073788:dctD protein, Rhizobium meliloti