DNA conformational changes associated with the cooperative binding of cI-repressor of bacteriophage λ to O_R

The cI repressor protein (cI) maintains bacteriophage λ in the lysogenic state in infected Escherichia coli cells by binding cooperatively to three tandemly repeated sequences comprising the right operator (O_R). Cooperative interactions occur between alternate pairs of cI dimers bound to adjacent sites. Although crystallographic studies have revealed the structure of the DNA in the 92 amino acid residue amino-terminal fragment-O_LI complex, the structure of the DNA within the O_R-cI complex with intact, cooperatively bound cI has not been described. In this study, the structure of the DNA within O_R was quantitatively examined using sequence and structure-dependent nuclease cleavage patterns as a function of cI binding. The cooperative binding of cI to O_R1 and O^R2 induces a conformational change in the DNA of O_R3 that is detectable by both DNase I and 5-phenyl-1,10-phenanthroline. Hydroxyl radical footprinting indicates the presence of an "A-tract" between O_R1 and O_R2 at the site of a run of four adenine-thymine base-pairs, implying a stable bend between the sites of approximately 18°. 5-Phenyl-1,10-phenanthroline footprinting reports conformational changes within the central base-pairs of all three sites that is dependent upon the sequence-specific binding of cI. The observed conformational changes are more extensive within O_R2 and O_R3 compared with O_R1, consistent with an "induced-fit" model of sequence-specific recognition. A number of changes in nuclease reactivity within the individual binding sites were quantitatively correlated with cI binding at the other sites within O_R. These results demonstrate that changes in the DNA structure are propagated among the sites in response to the binding of cI and imply a role for DNA sequence-dependent conformational changes in the mechanisms of both the intrinsic and cooperative binding reactions of cI to O_R.