Efficient and exclusive induction of Tet repressor by the oligopeptide Tip results from co-variation of their interaction site.
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.
Klotzsche
Marcus
M
Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Goeke
Dagmar
D
Berens
Christian
C
Hillen
Wolfgang
W
D019943:Amino Acid Substitution; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics
D010455:Peptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C521715:pepBs1-Ac peptide; C034811:tetracycline resistance-encoding transposon repressor protein; D013879:Thioredoxins