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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 the TetR variants by tetracycline (tc), anhydro-tetracycline (atc) and doxycycline (dox). β-Gal activity was determined in the E. coli screening strain with TetR variants encoded by pWH527-derivatives. β-Gal activities are given in Miller Units (MU) with the maximal expression in the absence of TetR (w/o TetR, black bar). White bars show the expression levels obtained with the TetR variant indicated at the bottom. Light gray bars indicate the β-Gal activities after induction with 0.2 μg/ml tc, gray bars after induction with 0.2 μg/ml atc and dark gray bars after induction with 0.2 μg/ml dox.
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Figure 6: Induction of the TetR variants by tetracycline (tc), anhydro-tetracycline (atc) and doxycycline (dox). β-Gal activity was determined in the E. coli screening strain with TetR variants encoded by pWH527-derivatives. β-Gal activities are given in Miller Units (MU) with the maximal expression in the absence of TetR (w/o TetR, black bar). White bars show the expression levels obtained with the TetR variant indicated at the bottom. Light gray bars indicate the β-Gal activities after induction with 0.2 μg/ml tc, gray bars after induction with 0.2 μg/ml atc and dark gray bars after induction with 0.2 μg/ml dox.

Mentions: The TetR residues N82 and F86 contact tc as derived from the crystal structure of the TetR-tc complex (30). Hence, we tested whether the three TetR mutants in which one or both of these residues are replaced by A are inducible by tc, atc or dox, three widely used inducers of TetR. The results are shown in Figure 6. TetR-F86A is not induced by tc, but atc and dox are very efficient inducers. In contrast, TetR-N82A and TetR-N82A-F86A are not induced by any of these compounds. Since these two are best induced by Tip we have created functionally novel TetR mutants, which will not respond to the most commonly used inducers atc and dox anymore, but instead are highly sensitive to Tip.Figure 6.


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 the TetR variants by tetracycline (tc), anhydro-tetracycline (atc) and doxycycline (dox). β-Gal activity was determined in the E. coli screening strain with TetR variants encoded by pWH527-derivatives. β-Gal activities are given in Miller Units (MU) with the maximal expression in the absence of TetR (w/o TetR, black bar). White bars show the expression levels obtained with the TetR variant indicated at the bottom. Light gray bars indicate the β-Gal activities after induction with 0.2 μg/ml tc, gray bars after induction with 0.2 μg/ml atc and dark gray bars after induction with 0.2 μg/ml dox.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Induction of the TetR variants by tetracycline (tc), anhydro-tetracycline (atc) and doxycycline (dox). β-Gal activity was determined in the E. coli screening strain with TetR variants encoded by pWH527-derivatives. β-Gal activities are given in Miller Units (MU) with the maximal expression in the absence of TetR (w/o TetR, black bar). White bars show the expression levels obtained with the TetR variant indicated at the bottom. Light gray bars indicate the β-Gal activities after induction with 0.2 μg/ml tc, gray bars after induction with 0.2 μg/ml atc and dark gray bars after induction with 0.2 μg/ml dox.
Mentions: The TetR residues N82 and F86 contact tc as derived from the crystal structure of the TetR-tc complex (30). Hence, we tested whether the three TetR mutants in which one or both of these residues are replaced by A are inducible by tc, atc or dox, three widely used inducers of TetR. The results are shown in Figure 6. TetR-F86A is not induced by tc, but atc and dox are very efficient inducers. In contrast, TetR-N82A and TetR-N82A-F86A are not induced by any of these compounds. Since these two are best induced by Tip we have created functionally novel TetR mutants, which will not respond to the most commonly used inducers atc and dox anymore, but instead are highly sensitive to Tip.Figure 6.

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