Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions.

The transposon Tn10-encoded TET repressor controls the expression of tetracycline resistance as well as its own synthesis. The antibiotic tetracycline functions as an inducer for both genes, which are transcribed in divergent directions from a common start area. The interaction of the TET repressor with the regulatory sequence of the tetracycline resistance operon is investigated by equilibrium and kinetic methods. The wild-type control sequence contains two nearly identical operators separated by only ten base-pairs. A deletion mutant lacking one of the operators is constructed by controlled digestion with exonuclease Bal31. It serves to prove that the two TET operators are each occupied by a TET repressor dimer in the wild-type tet operon regulatory sequence. The association constants are approximately identical for both operators between 10(12) and 10(13) M-1 as derived from kinetic data. The half-life of the TET repressor--tet operator complex is 12 minutes when competed with tet operator DNA and two minutes when competed with the inducer tetracycline. The dissociation of the repressor--operator complex has no apparent activation enthalpy but has an activation entropy of -320 J/mol K, indicating the involvement of solvent or counterion condensation. The dissociation rate constant of the tetracycline--TET repressor complex depends strongly on temperature. The activation enthalpy is 160 kJ/mol, indicating extremely strong binding of the drug. This result is discussed with respect to the necessary sensitivity of a regulated resistance gene. The native structure of the TET repressor is a dimer, as demonstrated by molecular exclusion chromatography. The elution behavior of the TET repressor--tetracycline complex indicates clearly that the repressor--inducer complex remains a dimer. The results are discussed with respect to the regulatory functions of the components.