Flanking DNA-sequences contribute to the specific binding of cl-repressor and O_RI

The binding of cl-repressor to a series of mutant operators containing O_R1 of the right operator of bacteriophage lambda was investigated. Sites O_R2 and/or O_R3 were inactivated by either point or deletion mutations. The free energy of binding repressor to O_R1 in the wildtype operator, △G₁, is -13.7 ± 0.3 kcal/mol. △G₁, determined for an O_R2- operator created by a single point mutation in O_R2 is -13.6 ± 0.2 kcal/mol. In contrast, △G₁, for the binding of repressor to a cloned synthetic O_R1 operator containing only 24 bp of lambda sequence is -12.2 ± 0.1 kcal/mol. When sequence 5' to O_R1 is present, the binding affinity increases to -13.0 ± 0.1 kcal/mol. In addition, the proximity of O_R1 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 O_R1 binding-site contributes to the specific binding of cl-repressor.