Cooperative binding of DctD to the dctA upstream activation sequence of Rhizobium meliloti is enhanced in a constitutively active truncated mutant

DctD, a σ⁵⁴-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 ΔG(A)⁰ = -9.5 ± 0.3, ΔG(B)⁰ = -11.2 ± 0.2, and ΔG(AB)⁰ = -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 ΔG(A)⁰ = -9.4 ± 0.2, ΔG(B((G→C) = -10.0 ± 0.2, and ΔG(AB(G→C) = -2.2 ± 0.4. We previously showed that removing the two-component receiver domain from DctD, making DctD(Δ)(₁-₁₄₂), yields a constitutively active truncated protein. Global analysis of binding data for DctD(Δ)(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 (ΔG(A)⁰ = -9.4 ± 0.6, ΔG(B)⁰ = -11.1 ± 0.3, and ΔG(AB)⁰ = -3.8 ± 0.6.).