We report measurements of the relative binding affinity of CAP for DNA sequences which have been systematically mutated in the region flanking the consensus binding site. Our experiments focus on the locus one helical turn from the dyad axis where DNA bending toward the minor groove is induced upon C-AP binding. The binding free energy and extent of bending are moderately well correlated for the set of 56 sequences. Changes in binding affinity spanning a factor of about 50 could be accounted for by additive contributions of dinucleotides; with a few exceptions, the relative ranking of dinucleotide contributions to binding and bending are similar. We conclude that dinucleotides are the smallest independent unit required for quantitative interpretation of CAP-induced DNA bending and binding in the distal domains of the CAP consensus binding site. The imperfect correlation between binding strength and extent of bending implies that sequence changes affect protein binding strength not only by altering the DNA deformation energy required to form the complex, but also by affecting directly the free energy of interaction between protein and DNA.
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