Thermodynamic analysis of tetracycline-mediated induction of Tet repressor by a quantitative methylation protection assay

Abstract

We describe a method for quantitative detection and thermodynamic interpretation of tetracycline (tc)-mediated induction of the Tn10 encoded Tet repressor (TetR). Binding of dimeric TetR to the tet operator (tetO) was quantitated by protection of DNA from methylation as a function of te concentration. A thermodynamic scheme covering all single reactions relevant for TetR induction was used to interpret the data. The equilibrium association constants of the TetR-[Mg-tc]+ and TetR-[Mg-tc]2+ complexes to tetO were determined at different NaCl and TetR concentrations. Variation of total TetR concentration from 0.2 to 1.1 x 10(-7) M yielded identical results. A strong salt dependency of TetR-tetO binding was verified between 2.5 and 100 mM NaCl, whereas [Mg-tc]+ binding to TetR is independent of the ionic strength. The TetR-tetO binding constant drops 10(2)- to 10(3)-fold upon binding of the first and further 10(4)- to 10(7)-fold by binding of the second [Mg-tc]+. This apparent cooperativity of tc-mediated induction indicates that each [Mg-tc]+ interacts with both TetR monomers.