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Abstract

The recognition of operator DNA by Tet repressor was analyzed by fluorescence stopped flow measurements. The main part of the fluorescence change observed for the reaction of the repressor with operator DNA reflects a second-order binding reaction including the expected concentration dependence. Global fitting of transients measured at different concentrations reveal at least one intramolecular step in addition to the bimolecular step. The rate constant for the bimolecular step is strongly salt dependent approaching the limit of diffusion control 2×108 M–1 s–1 at 50 mM NaCl and decreasing to 5×104 M–1 s–1 at 600 mM NaCl. These data are consistent with initial formation of a pre-equilibrium complex; electrostatic steering resulting from the high dipole moment of the repressor may contribute to the strong salt dependence. The rate constants of the intramolecular step are in the range of 0.1 s–1. Fluorescence quenching is salt dependent; the overall binding constant to operator O1 at 150 mM NaCl is 5×108 M–1; binding constants at different salt concentrations indicate 5 ion contacts for the specific complex of the Tet repressor dimer. The binding constant to operator O2 is higher than to O1 by a factor of 2 at 400 mM NaCl.