Structural and thermodynamic linkages associated with cI repressor binding to the right operator of bacteriophage lambda

Abstract

The cI repressor protein (cI) maintains bacteriophage {dollar}\lambda{dollar} in the lysogenic state in infected E. coli cells by binding cooperatively to the three tandemly repeated sequences of the right operator (O{dollar}\sb{lcub}\rm R{rcub}{dollar}) of the phage {dollar}\lambda{dollar} genome. Cooperative interactions occur between alternate pairs of cI dimers bound to adjacent sites. The site-specific DNA binding activity has been localized to the amino terminal domain while the cooperative interactions are mediated primarily by the carboxyl terminal domains.;In these studies, the intrinsic binding energy of cI to O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 was altered in a defined manner through a series of mutations resulting in graded alterations in the binding energy. The free energy terms describing the binding of cI to these mutants operators were determined. The cooperative interaction energy between sites O{dollar}\sb{lcub}\rm R{rcub}{dollar}2 and O{dollar}\sb{lcub}\rm R{rcub}{dollar}3 is linked to the presence of a competent O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 site; little or no cooperativity is observed between O{dollar}\sb{lcub}\rm R{rcub}{dollar}2 and O{dollar}\sb{lcub}\rm R{rcub}{dollar}3 unless binding to the O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 site is completely eliminated. Mutations in the non-consensus half-site of O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 appear to lower the cooperative interaction energy between sites O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 and O{dollar}\sb{lcub}\rm R{rcub}{dollar}2, presumably by inhibiting formation of a structure facilitating cooperative interactions between the sites.;The structure of O{dollar}\sb{lcub}\rm R{rcub}{dollar} was examined using various sequence-dependent and structure-dependent cleavage probes. cI bound cooperatively to O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 and O{dollar}\sb{lcub}\rm R{rcub}{dollar}2 induces a DNase I hypersensitivity in O{dollar}\sb{lcub}\rm R{rcub}{dollar}3. 5-phenyl-1,10-phenanthroline footprinting reports structural changes involving alterations in sites O{dollar}\sb{lcub}\rm R{rcub}{dollar}2 and OR3 associated with cI binding, but no alterations in O{dollar}\sb{lcub}\rm R{rcub}{dollar}1. Hydroxyl radical footprinting indicates the presence of an "A-tract" between O{dollar}\sb{lcub}\rm R{rcub}{dollar}1 and O{dollar}\sb{lcub}\rm R{rcub}{dollar}2 at the site of a run of 4 adenines on one strand, implying a stable bend between sites of approximately 18{dollar}\sp\circ{dollar}. A variety of changes in cleavage observed with each reagent suggests that structural alterations are propagated between sites in response to binding of cI repressor. These results imply a role for sequence-dependent structure in both the intrinsic and cooperative interactions describing the binding of cI repressor to O{dollar}\sb{lcub}\rm R{rcub}{dollar}.