In this article we report the in vivo and in vitro characterization of single chain tetracycline repressor (scTetR) variants in Escherichia coli. ScTetR is genetically and proteolytically stable and exhibits the same regulatory properties as dimeric TetR in E.coli. Urea-dependent denaturation of scTetR is independent of the protein concentration and follows the two-state model with a monophasic transition. Contrary to dimeric TetR, scTetR allows the construction of scTetR mutants, in which one subunit contains a defective inducer binding site while the other is functional. We have used this approach to establish that scTetR needs occupation of both inducer binding sites for in vivo and in vitro induction. Single mutations causing loss of induction in dimeric TetR lead to non-inducible scTetR when inserted into one half-side. The construction of scTetR H64K S135L S138I (scTetRi2) in which one half-side is specific for 4-dedimethylamino-anhydrotetracycline (4-ddma-atc) and the other for tetracycline (tc) leads to a protein which is only inducible by the mixture of tc and 4-ddma-atc. Fluorescence titration of scTetRi2 with both inducers revealed distinct occupancy with each of these inducers yielding roughly a 1:1 stoichiometry of each inducer per scTetRi2. The properties of this gain of function mutant clearly demonstrate that scTetR requires the binding of two inducers for induction of transcription.

Tet repressor mutants with a shifted effector specificity, preference for a mutant operator sequence or reversion of activity were combined to construct variants bearing two or three phenotypic alterations. TetR alleles with combinations of altered operator and effector specificities can be created by merging the respective residues in a single polypeptide. The mutations giving rise to revTetR, on the other hand, show drastic influences on the ligand binding phenotypes when combined with respective alterations. One TetR variant displays all three phenotypic alterations and thus demonstrates the general possibility of implementing them in one protein.