Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor.
We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant K(A) of revTetR for binding of [atcMg](+) is approximately 10(8) M(-1), four orders of magnitude lower than that of TetR. The affinity of TetR for tetO is 5.6 +/- 2 x 10(9) M(-1) and that for revTetR in the presence of atc is 1 +/- 0.2 x 10(8) M(-1). Both induced forms, the atc-bound TetR and the free revTetR, have the same low affinity of 4 +/- 1 x 10(5) M(-1) for DNA. Therefore, atc does not act as a dimerization agent for revTetR. We discuss the structural differences between TetR and revTetR potentially underlying this reversal of activity.
Kamionka
Annette
A
Lehstuhl für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.
Bogdanska-Urbaniak
Joanna
J
Scholz
Oliver
O
Hillen
Wolfgang
W
D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009875:Operator Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013754:Tetracyclines / Q000494:pharmacology*; D013816:Thermodynamics
D004269:DNA, Bacterial; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; C016229:4-epianhydrotetracycline