DNA CONFORMATIONAL-CHANGES ASSOCIATED WITH THE COOPERATIVE BINDING OFCI-REPRESSOR OF BACTERIOPHAGE-LAMBDA TO O-R

The cI repressor protein (cI) maintains bacteriophage lambda in the ly sogenic state in infected Escherichia coli. cells by binding cooperati vely to three tandemly repeated sequences comprising the right operato r (O-R) Cooperative interactions occur between alternate pairs of cI d imers bound to adjacent sites. Although crystallographic studies have revealed the structure of the DNA in the 92 amino acid residue amino-t erminal fragment-O(L)1 complex, the structure of the DNA within the O- R-cI complex with intact, cooperatively bound cI has not been describe d. In this study, the structure of the DNA within O-R was quantitative ly examined using sequence and structure-dependent nuclease cleavage p atterns as a function of cI binding. The cooperative binding of cI to O(R)1 and O(R)2 induces a conformational change in the DNA of O(R)3 th at is detectable by both DNase I and 5-phenyl-1, 10-phenanthroline. Hy droxyl radical footprinting indicates the presence of an ''A-tract'' b etween O(R)1 and O(R)2 at the site of a run of four adenine-thymine ba se-pairs, implying a stable bend between the sites of approximately 18 degrees. 5-Phenyl-1,10-phenanthroline footprinting reports conformati onal changes within the central base-pairs of all three sites that is dependent upon the sequence-specific binding of cI. The observed confo rmational changes are more extensive within O(R)2 and O(R)3 compared w ith O(R)1, consistent with an ''induced-fit'' model of sequence-specif ic recognition. A number of changes in nuclease reactivity within the individual binding sites were quantitatively correlated with cI bindin g at the other sites within O-R. These results demonstrate that change s in the DNA structure are propagated among the sites in response to t he binding of cI and imply a role for DNA sequence-dependent conformat ional changes in the mechanisms of both the intrinsic and cooperative binding reactions of cI to O-R.