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

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Abstract
The binding of cI-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, .DELTA.G1, is -13.7 .+-. 0.3 kcal/mol. .DELTA.G1 determined for an OR2- operator created by a single point mutation in OR2 is -13.6 .+-. 0.2 kcal/mol. In contrast, .DELTA.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 .DELTA.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 cI-repressor.