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Efficient and exclusive induction of Tet repressor by the oligopeptide Tip results from co-variation of their interaction site.

Klotzsche M, Goeke D, Berens C, Hillen W - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

ABSTRACT
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.

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Induction of TetR and TetR-N82A by C-terminal SbmC-Tip fusions. Induction of the TetR variants indicated above the plot and encoded by pWH527-derivatives is given by the β-Gal activities expressed by the screening strain. Gray bars represent basal expression levels of SbmC(C)-Tip fusions in the absence of IPTG, while induced (60 µM IPTG) expression levels are represented by black bars. The sequence of Tip fused to SbmC is illustrated below the chart. The residues of Tip fused to SbmC are shown in bold, and the linker between SbmC and Tip is shown in light print.
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Figure 4: Induction of TetR and TetR-N82A by C-terminal SbmC-Tip fusions. Induction of the TetR variants indicated above the plot and encoded by pWH527-derivatives is given by the β-Gal activities expressed by the screening strain. Gray bars represent basal expression levels of SbmC(C)-Tip fusions in the absence of IPTG, while induced (60 µM IPTG) expression levels are represented by black bars. The sequence of Tip fused to SbmC is illustrated below the chart. The residues of Tip fused to SbmC are shown in bold, and the linker between SbmC and Tip is shown in light print.

Mentions: The induction properties of TrxA-Tip could depend in part on the TrxA portion of the fusion protein. To address this question, we constructed Tip and MTip fusions to the C-terminus of the E. coli protein SbmC (27) and replaced the TrxA-Tip fusion in our screening strain by them. SbmC is a small, soluble protein of 157 residues with solvent-exposed and flexible N- and C-termini (28). It is part of the SOS regulon and involved in reducing DNA damage (29). Induction of TetR by SbmC(C)-MTip is slightly more efficient than induction by SbmC(C)-Tip. In contrast, TetR-N82A is induced to a 5-fold higher level of β-Gal activity by SbmC(C)-MTip compared to SbmC(C)-Tip as shown in Figure 4. This result establishes an only marginal influence of the carrier protein on Tip activity. Hence, the improved induction of the TetR variants by the elongated Tip results solely from their effects on Tip–TetR interaction.Figure 4.


Efficient and exclusive induction of Tet repressor by the oligopeptide Tip results from co-variation of their interaction site.

Klotzsche M, Goeke D, Berens C, Hillen W - Nucleic Acids Res. (2007)

Induction of TetR and TetR-N82A by C-terminal SbmC-Tip fusions. Induction of the TetR variants indicated above the plot and encoded by pWH527-derivatives is given by the β-Gal activities expressed by the screening strain. Gray bars represent basal expression levels of SbmC(C)-Tip fusions in the absence of IPTG, while induced (60 µM IPTG) expression levels are represented by black bars. The sequence of Tip fused to SbmC is illustrated below the chart. The residues of Tip fused to SbmC are shown in bold, and the linker between SbmC and Tip is shown in light print.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919500&req=5

Figure 4: Induction of TetR and TetR-N82A by C-terminal SbmC-Tip fusions. Induction of the TetR variants indicated above the plot and encoded by pWH527-derivatives is given by the β-Gal activities expressed by the screening strain. Gray bars represent basal expression levels of SbmC(C)-Tip fusions in the absence of IPTG, while induced (60 µM IPTG) expression levels are represented by black bars. The sequence of Tip fused to SbmC is illustrated below the chart. The residues of Tip fused to SbmC are shown in bold, and the linker between SbmC and Tip is shown in light print.
Mentions: The induction properties of TrxA-Tip could depend in part on the TrxA portion of the fusion protein. To address this question, we constructed Tip and MTip fusions to the C-terminus of the E. coli protein SbmC (27) and replaced the TrxA-Tip fusion in our screening strain by them. SbmC is a small, soluble protein of 157 residues with solvent-exposed and flexible N- and C-termini (28). It is part of the SOS regulon and involved in reducing DNA damage (29). Induction of TetR by SbmC(C)-MTip is slightly more efficient than induction by SbmC(C)-Tip. In contrast, TetR-N82A is induced to a 5-fold higher level of β-Gal activity by SbmC(C)-MTip compared to SbmC(C)-Tip as shown in Figure 4. This result establishes an only marginal influence of the carrier protein on Tip activity. Hence, the improved induction of the TetR variants by the elongated Tip results solely from their effects on Tip–TetR interaction.Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus