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Volume 271, Number 42, Issue of October 18, 1996 pp. 26435-26442
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Cooperative Binding of DctD to the dctA Upstream Activation Sequence of Rhizobium meliloti Is Enhanced in a Constitutively Active Truncated Mutant

(Received for publication, May 24, 1996, and in revised form, August 8, 1996)

Dean Scholl and B. Tracy Nixon

From the  Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

DctD, a ⁵⁴-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 G⁰_A = 9.5 ± 0.3, G⁰_B = 11.2 ± 0.2, and G⁰_AB = 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 G⁰_A = 9.4 ± 0.2, G⁰_B(GC) = 10.0 ± 0.2, and G⁰_AB(GC) = 2.2 ± 0.4. We previously showed that removing the two-component receiver domain from DctD, making DctD_(1-142), yields a constitutively active truncated protein. Global analysis of binding data for DctD_(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 (G⁰_A = 9.4 ± 0.6, G⁰_B = 11.1 ± 0.3, and G⁰_AB = 3.8 ± 0.6.).

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