Purification of the TET repressor and TET operator from the transposon Tn10 and characterization of their interaction.

The TET repressor for the TnlO-encoded tetracycline resistance gene has been purified to near-homogeneity. The TET repressor binds ['Hltetracycline with an apparent association constant greater than lo7 litersmol-'. The formation of the TET repressor * tetracycline complex is optimal between pH 7.5 and pH 12 and is reduced below pH 7.5. At pH 5.0, the TET repressor is inactive. The thermal stability extends up to 55 "C and drops sharply to zero between 55 "C and 65 "C. Specific binding of the TET repressor to the TET operator is demonstrated by the protection of a single recognition site for the restriction endonuclease HincII out of two sites on the respective DNA fragment. This function of the TET repressor is inhibited in the presence of 4 PM tetracycline. The location of the operator is determined within 100 base pairs by the lack of protection of other restriction sites. The interaction of the TET repressor with the TET operator is characterized in vitro using short, homogeneous DNA fragments containing the "0-encoded TET gene control sequences, which were cloned under reconstruction of the Eco RI sites into pVH51. These fragments contain the TET gene control elements and different parts of flanking sequences. The 187-base pair (bp) fragment was prepared in milligram amounts from multiple insertion plasmids and radioactively labeled by filling in the protruding Eco RI ends using [%IATP. The TET repressor binds the 187-bp fragment with an apparent association constant greater than IO7 liters-mol". The stoichiometry is 4 TET repressor molecules/l87-bp DNA. A 300-fold molar excess of a 180-bp fragment prepared from a Hue III digest of pVH51 does not compete with the 187-bp DNA for TET repressor binding. This result is interpreted as specific interaction of the TET repressor with the TET operator in the 187-bp fragment. This specific interaction is inhibited in the presence of tetracycline. The concentration dependence of the inhibition reveals that the association constant for the TET repressoretetracycline complex exceeds the one for the TET repressor TET operator complex. The thermal stability of the TET repressor TET operator complex extends up to 60 "C and drops sharply between 63 "C and 70 "C. The TET repressor-TET operator complex is stable be-