DNA Sequence Dependent and Independent Conformational Changes in Multipartite Operator Recognition by λ-Repressor

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Abstract
Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein−protein interaction, resulting in cooperative binding. The operators of bacteriophage λ have several pairs of repressor binding sites (OR1−OR2, OR2−OR3, OL1−OL2, and OL2−OL3) separated by a variable number of base pairs, and thus, bacteriophage λ is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in OR1−OR2 and OL2−OL3 but much less so in OR2−OR3. Upon titration of λ-repressor with single-operator sites OR1, OR2, and OR3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA−protein complexes. Acrylamide quenching of tryptophan fluorescence of λ-repressor bound to these single operators also shows different Stern−Volmer constants, supporting the above conclusions. Titration of λ-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by OR1−OR2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein−protein contact. Stern−Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of λ-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, OR2−OR3-bound repressor has significantly higher acrylamide quenchable components than either of the OR2- or OR3-bound proteins, again suggesting additional conformational changes upon establishment of protein−protein contact. We conclude that the strategy of recognition of multipartite operator by λ-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence.