Tet Repressor Induction without Mg2+

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Abstract
We have examined anhydrotetracycline (atc) binding to Tet repressor (TetR) in dependence of the Mg2+ concentration. Of all tc compounds tested so far, atc has the highest affinity for TetR, with a KA of 9.8 × 1011 M-1 in the presence of Mg2+ and 6.5 × 107 M-1 without Mg2+. Thus, it binds TetR with 500-fold higher affinity than tc under both conditions. The Mg2+-free binding of atc to TetR leads to induction in vitro, demonstrating that the metal is not necessary to trigger the associated conformational change. To obtain more detailed information about Mg2+-free induction, we constructed and prepared to homogeneity four single-alanine substitution mutants of TetR. Three of them affect residues involved in contacting Mg2+ (TetR H100A, E147A, and T103A), and one altered residue contacts tc TetR N82A. TetR H100A and E147A are induced by atc, with and without Mg2+, showing 110-fold and 1000-fold decreased Mg2+-dependent and unchanged Mg2+-independent atc binding, respectively. Thus, the contacts of these residues to Mg2+ are not necessary for induction. TetR N82A is not inducible under any of the conditions employed and shows an about 4000-fold decreased atc binding constant. The Mg2+-dependent affinity of TetR T103A for atc is only 400-fold reduced, but no induction with atc was observed. Thus, Thr103 must be essential for the conformational change associated with induction in the absence of Mg2+.