Kinetics and Equilibrium Studies of Tet Repressor–Operator Interaction

Binding of a Tet repressor mutant containing a single Trp43 residue in the tet operator recognition α-helix leads to the quenching of the protein fluorescence down to about 23% in the case of the tet O1 operator and to 40% in the case of the tet O2 operator. We have used fluorescence detection to describe the binding equilibrium and kinetics of the Tet repressor interaction with the 20-bp DNA operators tet O1 and tet O2. Stopped-flow measurements in an excess of the tet operators performed in 5 mM NaCl or 150 mM NaCl indicate that the reaction can be described by at least three exponentials characterized by different relaxation times. The mechanism of interaction for both operators as well as for two salt concentrations used can be described as TetR + Operator ⇔ Complex 1 ⇔ Complex 2 ⇔ Complex 3. Only the much faster process can be described as a second-order reaction characterized by a bimolecular rate constant equal to 2.8 × 106 M−1 sec−1 for both operators. The medium and slow processes may be described by relaxational times ranging from 50 msec to seconds. The results of the binding equilibrium measurements extrapolated to 1 M NaCl concentration, which reflects the specific nonionic interaction between TetR and tet operators, indicate Kas equal to 3.2 × 104 and 4.0 × 105 M−1 for tet O1 and tet O2, respectively. The number of monovalent ions replaced upon binding can be calculated as about 5 and 3 for tet O1 and tet O2, respectively. The binding of Tet repressor to the operators leads to changes in the circular dichroism spectra of the DNA which could indicate transitions of B-DNA into A-like DNA structure.