The binding of cl-repressor to a series of mutant operators containing OR1 of the right operator of bacteriophage lambda was investigated. Sites OR2 and/or OR3 were inactivated by either point or deletion mutations. The free energy of binding repressor to OR1 in the wildtype operator, △G1, is -13.7 ± 0.3 kcal/mol. △G1, determined for an OR2- operator created by a single point mutation in OR2 is -13.6 ± 0.2 kcal/mol. In contrast, △G1, for the binding of repressor to a cloned synthetic OR1 operator containing only 24 bp of lambda sequence is -12.2 ± 0.1 kcal/mol. When sequence 5' to OR1 is present, the binding affinity increases to -13.0 ± 0.1 kcal/mol. In addition, the proximity of OR1 to a fragment-end decreases △G1, from -13.7 to -12.3 ± 0.1 kcal/mol. These results suggest that the DNA sequence outside the 17 bp OR1 binding-site contributes to the specific binding of cl-repressor.
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