DNA sequence dependent and independent conformational changes in multipartite operator recognition by λ-repressor

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 (O_R1-O_R2, O_R2-O_R3, O_L1-O_L2, and O_L2-O_L3) 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 O_R1-O_R2 and O_L2-O_L3 but much less so in O_R2-O_R3. Upon titration of λ-repressor with single-operator sites O_R1, O_R2, and O_R3, 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 O_R1-O_R2 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, O_R2-O_R3-bound repressor has significantly higher acrylamide quenchable components than either of the O_R2- or O_R3-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.