Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage
have several pairsof repressor binding sites (O
R1-O
R2, O
R2-O
R3, O
L1-O
L2, and O
L2-O
L3) separated by a variable numberof base pairs, and thus, bacteriophage
is a model system for studying multipartite operator recognitionby 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
-repressor with single-operator sitesO
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. Acrylamidequenching of tryptophan fluorescence of
-repressor bound to these single operators also shows differentStern-Volmer constants, supporting the above conclusions. Titration of
-repressor with oligonucleotidescontaining 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 thesingle operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescenceof
-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 acrylamidequenchable components than either of the O
R2- or O
R3-bound proteins, again suggesting additionalconformational changes upon establishment of protein-protein contact. We conclude that the strategy ofrecognition of multipartite operator by
-repressor is complex and varied, involving conformational changesin both DNA and protein that are determined by the separation of the binding sites as well as the nucleicacid sequence.