AFFINITY AND SPECIFICITY OF TRP REPRESSOR-DNA INTERACTIONS STUDIED WITH FLUORESCENT OLIGONUCLEOTIDES

Fluorescence-based solution methods have been used to study the bindin g of the Erp repressor of Escherichia coli to a series of oligonucleot ides bearing all or partial determinants for high affinity specific bi nding. The tryptophan, salt concentration and competitor DNA dependenc e 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 (GAACTAGTTAACTAGT AC) and is known to bind repressor in a 1:1 dimer-DNA complex, resulte d 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 4 x 10(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 oi the hole -repressor to this oligonucleotide was approximately half as large as what would be predicted by the number of phosphate contacts in the cry stal 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-sit es GNACT separated by an eight base-pair spacer containing none of the natural intervening sequence. The rather low affinity observed for th e 36mer revealed that the intervening sequence in the natural operator s 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), regardles s of tryptophan concentration, whereas binding to sequences bearing pa rtial specificity ratio between 100 and 1000, depending upon the salt concentration. Even in abscence of added KCl, the specificity ratio of trp repressor was greater than 100, implicating a significant free en ergy contribution from non-electrostatic Interaction forces. (C) 1997 Academic Press Limited.