title: Induction of single chain tetracycline repressor requires the binding of two inducers.
abstract: 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 (scTetR(i2)) 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 scTetR(i2) with both inducers revealed distinct occupancy with each of these inducers yielding roughly a 1:1 stoichiometry of each inducer per scTetR(i2). The properties of this gain of function mutant clearly demonstrate that scTetR requires the binding of two inducers for induction of transcription.
authors.affiliation: Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Kamionka
authors.forename: Marius
authors.initials: M
authors.lastname: Majewski
authors.forename: Karin
authors.initials: K
authors.lastname: Roth
authors.forename: Ralph
authors.initials: R
authors.lastname: Bertram
authors.forename: Christine
authors.initials: C
authors.lastname: Kraft
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D013752:Tetracycline / Q000378:metabolism / Q000494:pharmacology; D013754:Tetracyclines / Q000378:metabolism / Q000494:pharmacology; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline