Nucleic Acids Research Advance Access published online on June 1, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm357
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Molecular Biology |
Efficient and exclusive induction of Tet repressor by the oligopeptide Tip results from co-variation of their interaction site
Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany
*To whom correspondence should be addressed. Tel: +49 9131 8528081; Fax: +49 9131 8528082; Email: whillen{at}biologie.uni-erlangen.de
Received February 22, 2007. Revised April 20, 2007. Accepted April 24, 2007.
Protein–protein interactions are an important element of signal transfer within and between organisms. They are mainly mediated by short oligopeptide motifs and represent a widely used alternative to small, organic molecules for conveying information. The transcription factor TetR, a regulator of tetracycline resistance in Gram-negative bacteria, is naturally induced by tetracycline or its derivatives. The oligopeptide Tip (Transcription inducing peptide) fused to either N- or C-terminus of Thioredoxin A (TrxA) has been isolated as an artificial inducer for TetR in Escherichia coli. This inducing property can be exploited to monitor the in vivo expression of a protein of interest by fusing Tip to its C-terminus. We improve the induction efficiency of Tip by adding an aromatic amino acid before residue 1 of Tip in C-terminal fusions to TrxA. The induction efficiency of that modified TrxA-Tip fusion is further enhanced when the effector-binding pocket of TetR is enlarged by the N82A and F86A mutations. The double mutant is also insensitive to induction by tetracyclines. Thus, Tip is an exclusive inducer of this TetR variant, representing the first example of fully converting a small molecular weight effector-dependent transcription factor into one depending solely on protein–protein recognition.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.