COOPERATIVE BINDING OF DCTD TO THE DCTA UPSTREAM ACTIVATION SEQUENCE OF RHIZOBIUM-MELILOTI IS ENHANCED IN A CONSTITUTIVELY ACTIVE TRUNCATEDMUTANT

DctD, a sigma(54)-dependent, two-component regulator, binds to promote r distal (A) and promoter proximal (B) sites in an activation sequence located upstream of the dctA promoter. We report gel filtration and q uantitative DNase I footprint experiments supporting a model in which DctD(2) binds to these sites cooperatively, The global analysis of ups tream 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 Delta G(A)(0) = -9.5 +/- 0.3 , Delta G(B)(0) = -11.2 +/- 0.2, and Delta G(AB)(0) = -2.5 +/- 0.5. A separate analysis of data from upstream activation sequences containin g site A and a point mutant of site B, and site A and mutant site B on e-half helical turn out of phase confirmed the estimate of cooperativi ty, yielding free energy values of Delta G(A)(0) = -9.4 +/- 0.2, Delta G(B(G-->C))(0) = -10.0 +/- 0.2, and Delta G(AB(G-->C))(0) = -2.2 +/- 0.4. We previously showed that removing the two-component receiver dom ain from DctD, making DctD(Delta(1-142)), yields a constitutively acti ve truncated protein. Global analysis of binding data for DctD(Delta(1 -142)) showed that this constitutively active mutant has intrinsic bin ding energies equal to that of the inactive DctD protein, but that it displays significantly higher cooperativity (Delta G(A)(0) = -9.4 +/- 0.6, Delta G(B)(0) = -11.1 +/- 0.3, and Delta G(AB)(0) = -3.8 +/- 0.6. ).