Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides.

Fluorescence-based solution methods have been used to study the binding of the trp repressor of Escherichia coli to a series of oligonucleotides bearing all or partial determinants for high affinity specific binding. The tryptophan, salt concentration and competitor DNA dependence of the binding affinities was examined for these targets. Binding to a fluorescein-labeled 20 base-pair hairpin structure oligonucleotide, which contains a palindromic repressor binding site (GAACTAGTTAACTAGTAC) and is known to bind repressor in a 1 : 1 dimer-DNA complex, resulted in a protein concentration-dependent, competable static quenching of fluorescence in presence of co-repressor, l-tryptophan. The affinity recovered from the fits of these intensity profiles at 100 mM KCl was on the order of 4x10(8) M-1. In absence of co-repressor an increase in intensity at high repressor concentration (>10(-7) M) was observed. The salt concentration dependence of the specific binding of the holo-repressor to this oligonucleotide was approximately half as large as what would be predicted by the number of phosphate contacts in the crystal structures of the complex. Repressor binding to the fluorescein-labeled hairpin 20mer was compared with binding to a rhodamine-labeled 36 base-pair oligonucleotide bearing two inverted structural half-sites GNACT separated by an eight base-pair spacer containing none of the natural intervening sequence. The rather low affinity observed for the 36mer revealed that the intervening sequence in the natural operators contains energetic specificity determinants. Binding to a rhodamine-labeled oligonucleotide bearing a completely non-specific sequence was shown to occur over the same concentration range (>100 nM), regardless of tryptophan concentration, whereas binding to sequences bearing partial specificity ratio between 100 and 1000, depending upon the salt concentration. Even in absence of added KCl, the specificity ratio of trp repressor was greater than 100, implicating a significant free energy contribution from non-electrostatic interaction forces.