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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 mutants by Tip variants. Induction of TetR mutants by basal expression levels (absence of IPTG) of TrxA-XTip variants is shown. X represents any of the 20 amino acids as depicted in one-letter abbreviations (see upper right insert for the sequence). TetR variants are encoded by pWH527-derivatives. Induction of TetR variants is indicated by the corresponding β-Gal activities expressed by the screening strain. White bars show the induction values of TetR-N82A, gray bars those of TetR-F86A and black bars those of TetR-N82A-F86A. Induction by the initially analysed M insertion is highlighted by a black box. Tip indicates induction by basal expression levels of TrxA(C)-Tip and the one-letter abbreviations indicate the inserted residue. The insert in the upper left corner shows the coding of the bars. The central insert shows Western blots indicating the steady-state protein levels of TetR, TetR-N82A, TetR-F86A and TetR-N82A-F86A. The molecular mass of the TetR variants is indicated on the left.
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Figure 5: Induction of TetR mutants by Tip variants. Induction of TetR mutants by basal expression levels (absence of IPTG) of TrxA-XTip variants is shown. X represents any of the 20 amino acids as depicted in one-letter abbreviations (see upper right insert for the sequence). TetR variants are encoded by pWH527-derivatives. Induction of TetR variants is indicated by the corresponding β-Gal activities expressed by the screening strain. White bars show the induction values of TetR-N82A, gray bars those of TetR-F86A and black bars those of TetR-N82A-F86A. Induction by the initially analysed M insertion is highlighted by a black box. Tip indicates induction by basal expression levels of TrxA(C)-Tip and the one-letter abbreviations indicate the inserted residue. The insert in the upper left corner shows the coding of the bars. The central insert shows Western blots indicating the steady-state protein levels of TetR, TetR-N82A, TetR-F86A and TetR-N82A-F86A. The molecular mass of the TetR variants is indicated on the left.

Mentions: We wondered if the induction efficiency profiles of the TrxA(C)-XTip variants depend on the shape of the inducer-binding pocket. In addition to the TetR-N82A and -F86A mutants, we constructed the double mutant TetR-N82A-F86A and introduced that also into the screening strain. The Western blot shown as insert in Figure 5 indicates that TetR and TetR-F86A are expressed to about the same level, while TetR-N82A and TetR-N82A-F86A are present in roughly half the amount of the former variants. The induction activities were scored without IPTG at the basal expression levels of the TrxA(C)-XTip variants to increase the sensitivity and are shown in Figure 5. TrxA(C)-Tip shows about the same induction for TetR-N82A and TetR-F86A (∼2% β-Gal activity), but the double mutant appears slightly de-repressed under these conditions (∼4%). No other TrxA(C)-XTip variant induces TetR-N82A to a significantly higher degree than TrxA-MTip, and only TrxA(C)-FTip and TrxA(C)-WTip reach about the same induction efficiency (see the white columns in Figure 5). In contrast, TetR-F86A is very efficiently induced by TrxA-XTip variants with an aromatic residue. While TrxA-MTip shows only ∼6% β-Gal activity, TrxA(C)-HTip and TrxA(C)-YTip lead to ∼35% β-Gal activity, TrxA(C)-WTip gives rise to 53% and TrxA-FTip even to 60% β-Gal activity. The latter variant is hence the most efficient inducer of TetR-F86A. Similar to TetR-F86A, we observed the highest induction activities for TetR-N82A-F86A by TrxA-XTip derivatives with aromatic residues, albeit in a slightly changed order: TrxA-YTip exhibits the same induction efficiency for both TetR variants, while TrxA-HTip, TrxA-FTip and TrxA-WTip are better inducers for the double than for the single TetR mutant. Taken together, it appears that TrxA(C)-FTip and TrxA(C)-WTip are the best inducers for all three TetR variants, but their efficiencies increase from TetR-N82A via TetR-F86A to TetR-N82A-F86A. It is also noteworthy to mention that these induction profiles do not correlate generally to the differences in expression levels established for these three TetR variants.Figure 5.


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 mutants by Tip variants. Induction of TetR mutants by basal expression levels (absence of IPTG) of TrxA-XTip variants is shown. X represents any of the 20 amino acids as depicted in one-letter abbreviations (see upper right insert for the sequence). TetR variants are encoded by pWH527-derivatives. Induction of TetR variants is indicated by the corresponding β-Gal activities expressed by the screening strain. White bars show the induction values of TetR-N82A, gray bars those of TetR-F86A and black bars those of TetR-N82A-F86A. Induction by the initially analysed M insertion is highlighted by a black box. Tip indicates induction by basal expression levels of TrxA(C)-Tip and the one-letter abbreviations indicate the inserted residue. The insert in the upper left corner shows the coding of the bars. The central insert shows Western blots indicating the steady-state protein levels of TetR, TetR-N82A, TetR-F86A and TetR-N82A-F86A. The molecular mass of the TetR variants is indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Induction of TetR mutants by Tip variants. Induction of TetR mutants by basal expression levels (absence of IPTG) of TrxA-XTip variants is shown. X represents any of the 20 amino acids as depicted in one-letter abbreviations (see upper right insert for the sequence). TetR variants are encoded by pWH527-derivatives. Induction of TetR variants is indicated by the corresponding β-Gal activities expressed by the screening strain. White bars show the induction values of TetR-N82A, gray bars those of TetR-F86A and black bars those of TetR-N82A-F86A. Induction by the initially analysed M insertion is highlighted by a black box. Tip indicates induction by basal expression levels of TrxA(C)-Tip and the one-letter abbreviations indicate the inserted residue. The insert in the upper left corner shows the coding of the bars. The central insert shows Western blots indicating the steady-state protein levels of TetR, TetR-N82A, TetR-F86A and TetR-N82A-F86A. The molecular mass of the TetR variants is indicated on the left.
Mentions: We wondered if the induction efficiency profiles of the TrxA(C)-XTip variants depend on the shape of the inducer-binding pocket. In addition to the TetR-N82A and -F86A mutants, we constructed the double mutant TetR-N82A-F86A and introduced that also into the screening strain. The Western blot shown as insert in Figure 5 indicates that TetR and TetR-F86A are expressed to about the same level, while TetR-N82A and TetR-N82A-F86A are present in roughly half the amount of the former variants. The induction activities were scored without IPTG at the basal expression levels of the TrxA(C)-XTip variants to increase the sensitivity and are shown in Figure 5. TrxA(C)-Tip shows about the same induction for TetR-N82A and TetR-F86A (∼2% β-Gal activity), but the double mutant appears slightly de-repressed under these conditions (∼4%). No other TrxA(C)-XTip variant induces TetR-N82A to a significantly higher degree than TrxA-MTip, and only TrxA(C)-FTip and TrxA(C)-WTip reach about the same induction efficiency (see the white columns in Figure 5). In contrast, TetR-F86A is very efficiently induced by TrxA-XTip variants with an aromatic residue. While TrxA-MTip shows only ∼6% β-Gal activity, TrxA(C)-HTip and TrxA(C)-YTip lead to ∼35% β-Gal activity, TrxA(C)-WTip gives rise to 53% and TrxA-FTip even to 60% β-Gal activity. The latter variant is hence the most efficient inducer of TetR-F86A. Similar to TetR-F86A, we observed the highest induction activities for TetR-N82A-F86A by TrxA-XTip derivatives with aromatic residues, albeit in a slightly changed order: TrxA-YTip exhibits the same induction efficiency for both TetR variants, while TrxA-HTip, TrxA-FTip and TrxA-WTip are better inducers for the double than for the single TetR mutant. Taken together, it appears that TrxA(C)-FTip and TrxA(C)-WTip are the best inducers for all three TetR variants, but their efficiencies increase from TetR-N82A via TetR-F86A to TetR-N82A-F86A. It is also noteworthy to mention that these induction profiles do not correlate generally to the differences in expression levels established for these three TetR variants.Figure 5.

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