Changing the DNA-binding specificity of a repressor

The Mnt repressor of Salmonella phage P22 recognizes a 17 base pair operator. We constructed an Mnt binding site with two symmetric operator-constitutive mutations. We then selected Mnt mutants that have lost the ability to bind the wild-type operator but acquired the ability to bind the mutant operator. Four independent mutations change the same CAC codon in the mnt gene to CCT or CCC. The corresponding amino acid change in Mnt is His6 leads to Pro. DNA binding assays with purified wild-type and mutant proteins confirm the change of binding specificity in vitro. Wild-type Mnt binds strongly to the wild-type operator, but binds the mutant operator with 1000-fold less affinity. The mutant Mnt binds with converse affinities to the two operators.