The binding of Cro repressor to the synthetic operator DNA has been studied by systematic base substitutions. From this data we deduce specific interactions between Cro and the operator DNA and estimate the energetic contribution of each interaction to the specific binding. Such studies clearly show that the recognition of specific DNA sequences by Cro repressor is mediated by a combination of bi-dentate H-bonds between amino acid side chains and base pairs, and hydrophobic interactions with thymine's methyl groups as exposed within the DNA major groove. Losses of such H-bond or hydrophobic interactions reduce the binding free energy by 0.9 to 3.1 Kcal/mol or 0.8 to 1.6 Kcal/mol, respectively. The free energy changes are principally additive the specific binding, but not additive for nonspecific binding. These interactions described here are not only the specificity determinants in the sequence recognition, but also provide a large part of the binding free energy for the specific interaction of Cro repressor with DNA.
