THERMODYNAMIC ANALYSIS OF TETRACYCLINE-MEDIATED INDUCTION OF TET REPRESSOR BY A QUANTITATIVE METHYLATION PROTECTION ASSAY

We describe a method for quantitative detection and thermodynamic inte rpretation of tetracycline (tc)-mediated induction of the Tn10 encoded Tet repressor (TetR). Binding of dimeric TetR to the tet operator (te tO) was quantitated by protection of DNA from methylation as a functio n of tc concentration. A thermodynamic scheme covering all single reac tions 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 Tet R-tetO binding was verified between 2.5 and 100 mM NaCl, whereas [Mg-t c](+) binding to TetR is independent of the ionic strength. The TetR-t etO binding constant drops 10(2)- to 10(3)-fold upon binding of the fi rst and further 10(4)- to 10(7)-fold by binding of the second [Mg-tc]( +). This apparent cooperativity of tc-mediated induction indicates tha t each [Mg-tc](+) interacts with both TetR monomers. (C) 1995 Academic Press, Inc.