Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein

The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail th e interplay between DNA and protein sequence, we have examined binding of v ariant Inc operator DNA sequences to a series of mutant lactose regressor p roteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains . Variant operators differed from the wild-type operator, O-1, in spacing a nd/or symmetry of the half-sites that contact the LacI N-terminal DNA bindi ng domain. Binding of wild-type and mutant proteins was affected differenti ally by variations in operator sequence and symmetry. While the mutant seri es exhibits a 10(4)-fold range in binding affinity for O-1 operator, only a similar to 20-fold difference in affinity is observed for a completely sym metric operator, O-sym, used widely in studies of the LacI protein. Further , DNA sequence influenced allosteric response for these proteins. Binding o f this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pa ir sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence ma y establish the binding environment for protein and determine both affinity and potential for allostery.