Lambda repressor mutations that increase the affinity and specificity of operator binding

Intragenic, second-site reversion has been used to identify amino acid substitutions that increase the affinity and specificity of the binding of lambda repressor to its operator sites. Purified repressors bearing the second-site substitutions bind operator DNA from 3 to 600 fold more strongly than wild type; these affinity changes result from both increased rates of operator association and decreased rates of operator dissociation. Three of the revertant substitutions occur in the alpha 2 and alpha 3 DNA binding helices of repressor and seem to increase affinity by introducing new salt-bridges or hydrogen bonds with the sugar-phosphate backbone of the operator site. The fourth substitution alters the alpha 5 dimerization helix of repressor and appears to increase operator affinity indirectly.