P-31 NUCLEAR-MAGNETIC-RESONANCE SPECTRA AND DISSOCIATION-CONSTANTS OF LAC REPRESSOR HEADPIECE.DUPLEX OPERATOR COMPLEXES - THE IMPORTANCE OF PHOSPHATE ESTER BACKBONE FLEXIBILITY IN PROTEIN.DNA RECOGNITION

An alkaline phosphatase assay was used to determine the dissociation constants (K(D)) of the lac repressor N-terminal 56 amino acid fragment of the wild type and of a Y7I mutant complexed to 22 base pair (bp) wild-type and mutant symmetrical operator sequences. K(D)'s in 0.35 M monovalent salt ranged from 5.4 x 10(-8) M for the wild-type repressor-wild-type operator complex to greater-than-or-similar-to 1 x 10(-6) M for the wild-type repressor.nonspecific DNA complex. Mutant operators O2 (G5 --> A5 and C16 --> T16) and O4 (G5 --> C5 and C16 --> G16) bind nearly as tightly as the wild-type headpiece, while mutant O3 (A8 --> T8 and T13 --> A13) binds over 5-fold poorer. Operators O1, O2, and O4 bind ca. 10-fold poorer to the Y7I mutant headpiece. Operator O3 binds 2-fold poorer to the mutant headpiece. The temperature and salt dependence on the dissociation constants of wild-type headpiece binding to 22-bp operator support the conclusion that the headpiece contains the major DNA recognition portion of the protein and that electrostatics plays as important a role in the binding of operator to headpiece as it does in the whole repressor. The P-31 NMR spectra of shortened 14-bp wild-type and mutant symmetrical operators bound to the N-terminal 56-residue headpiece of the Y7I mutant repressor were compared to the spectra of the same operator bound to the wild-type repressor headpiece. These results are consistent with a recent proposal [Karslake, C., Botuyan, M. V., & Gorenstein, D. G. (1992) Biochemistry 31, 1849-18581 that specific, tight-binding operator.protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes. This retention of backbone torsional freedom in tight complexes is entropically favorable and provides a mechanism for protein discrimination of different operator binding sites.