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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 activity of the C-terminal Met insertion mutant TrxA-Mtip. (A) A schematic presentation of the E. coli screening strain for TetR inducing peptides is shown (10). Escherichia coli DH5α(λtet50) contains lacZ under Tet control on the chromosome. Derivatives of pWH527 or pWH1413 constitutively express LacI and the TetR variants. The TetR dimer is depicted by light gray ovals with circular DNA-binding domains. TetR controls transcription of lacZ. The peptide-expressing plasmids encode either TrxA (pWH2200) or SbmC (pWH2300) or the respective fusion protein (e.g. TrxA(C)-XTip is encoded by pWH2102-X) under control of Ptac. A TetR inducing thioredoxin-peptide fusion leads to β-Gal expression as indicated by the arrow. The respective resistance genes and origins of replication of the plasmids are indicated. (B) Sequences of Tip peptides (bold) fused C-terminal or N-terminal to TrxA and the linker between TrxA and Tip are shown in one-letter abbreviations. (C) β-Gal activities obtained with the indicated TrxA-Tip fusions in the presence and absence of IPTG are shown. Gray bars show the repressed states in the absence of IPTG. Expression of the Tip-fusion proteins was induced with 60 µM IPTG (black bars). (D) Western blots indicating the steady-state levels of Tip-fusion proteins in the presence of 60 µM IPTG. Molecular weights of marker proteins (not shown) are indicated on the left side.
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Figure 1: Induction activity of the C-terminal Met insertion mutant TrxA-Mtip. (A) A schematic presentation of the E. coli screening strain for TetR inducing peptides is shown (10). Escherichia coli DH5α(λtet50) contains lacZ under Tet control on the chromosome. Derivatives of pWH527 or pWH1413 constitutively express LacI and the TetR variants. The TetR dimer is depicted by light gray ovals with circular DNA-binding domains. TetR controls transcription of lacZ. The peptide-expressing plasmids encode either TrxA (pWH2200) or SbmC (pWH2300) or the respective fusion protein (e.g. TrxA(C)-XTip is encoded by pWH2102-X) under control of Ptac. A TetR inducing thioredoxin-peptide fusion leads to β-Gal expression as indicated by the arrow. The respective resistance genes and origins of replication of the plasmids are indicated. (B) Sequences of Tip peptides (bold) fused C-terminal or N-terminal to TrxA and the linker between TrxA and Tip are shown in one-letter abbreviations. (C) β-Gal activities obtained with the indicated TrxA-Tip fusions in the presence and absence of IPTG are shown. Gray bars show the repressed states in the absence of IPTG. Expression of the Tip-fusion proteins was induced with 60 µM IPTG (black bars). (D) Western blots indicating the steady-state levels of Tip-fusion proteins in the presence of 60 µM IPTG. Molecular weights of marker proteins (not shown) are indicated on the left side.

Mentions: Recently, a 16-mer peptide termed Tip (Transcription inducing peptide; Figure 1A) was isolated via phage display. Tip is not only able to bind specifically to its target, the bacterial transcription factor Tet repressor (TetR), but also triggers a conformational change in vivo normally induced by tetracyclines, the natural effectors of TetR (10). TetR is widely used to control regulation of gene expression in prokaryotes and, as a fusion protein with an activation domain, in eukaryotes (11), because it combines high specificity for its cognate DNA sequence (tetO) with extremely sensitive induction by tetracyclines (tc), especially the potent analogues doxycycline (dox) and anhydrotetracycline (atc) (12,13). Non-tc inducers of TetR, like Tip, may lead to alternative inducers when used as a scaffold for peptidomimetics to generate novel, small-molecule effectors. The recently published crystal structure of the TetR–Tip complex provides a molecular basis for such efforts (14). Another application of Tip is to fuse it to a protein of interest and use it to analyse target protein expression in vivo by monitoring a TetR-controlled reporter (15). Although a wide variety of tag-systems suitable for diverse applications exist, Tip is the only protein tag that allows quantitative in vivo analysis of protein expression. Mandatory for this application is a high efficiency of induction to ensure that low levels of Tip fusion proteins can be detected. We demonstrate here that addition of an aromatic residue between C-terminally fused Tip and TrxA along with mutations in the effector-binding pocket of TetR lead to a strong increase of reporter induction. Furthermore, the introduced TetR mutations render it insensitive to induction by tetracyclines, thereby creating exclusive specificity for Tip.Figure 1.


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 activity of the C-terminal Met insertion mutant TrxA-Mtip. (A) A schematic presentation of the E. coli screening strain for TetR inducing peptides is shown (10). Escherichia coli DH5α(λtet50) contains lacZ under Tet control on the chromosome. Derivatives of pWH527 or pWH1413 constitutively express LacI and the TetR variants. The TetR dimer is depicted by light gray ovals with circular DNA-binding domains. TetR controls transcription of lacZ. The peptide-expressing plasmids encode either TrxA (pWH2200) or SbmC (pWH2300) or the respective fusion protein (e.g. TrxA(C)-XTip is encoded by pWH2102-X) under control of Ptac. A TetR inducing thioredoxin-peptide fusion leads to β-Gal expression as indicated by the arrow. The respective resistance genes and origins of replication of the plasmids are indicated. (B) Sequences of Tip peptides (bold) fused C-terminal or N-terminal to TrxA and the linker between TrxA and Tip are shown in one-letter abbreviations. (C) β-Gal activities obtained with the indicated TrxA-Tip fusions in the presence and absence of IPTG are shown. Gray bars show the repressed states in the absence of IPTG. Expression of the Tip-fusion proteins was induced with 60 µM IPTG (black bars). (D) Western blots indicating the steady-state levels of Tip-fusion proteins in the presence of 60 µM IPTG. Molecular weights of marker proteins (not shown) are indicated on the left side.
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Related In: Results  -  Collection

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Figure 1: Induction activity of the C-terminal Met insertion mutant TrxA-Mtip. (A) A schematic presentation of the E. coli screening strain for TetR inducing peptides is shown (10). Escherichia coli DH5α(λtet50) contains lacZ under Tet control on the chromosome. Derivatives of pWH527 or pWH1413 constitutively express LacI and the TetR variants. The TetR dimer is depicted by light gray ovals with circular DNA-binding domains. TetR controls transcription of lacZ. The peptide-expressing plasmids encode either TrxA (pWH2200) or SbmC (pWH2300) or the respective fusion protein (e.g. TrxA(C)-XTip is encoded by pWH2102-X) under control of Ptac. A TetR inducing thioredoxin-peptide fusion leads to β-Gal expression as indicated by the arrow. The respective resistance genes and origins of replication of the plasmids are indicated. (B) Sequences of Tip peptides (bold) fused C-terminal or N-terminal to TrxA and the linker between TrxA and Tip are shown in one-letter abbreviations. (C) β-Gal activities obtained with the indicated TrxA-Tip fusions in the presence and absence of IPTG are shown. Gray bars show the repressed states in the absence of IPTG. Expression of the Tip-fusion proteins was induced with 60 µM IPTG (black bars). (D) Western blots indicating the steady-state levels of Tip-fusion proteins in the presence of 60 µM IPTG. Molecular weights of marker proteins (not shown) are indicated on the left side.
Mentions: Recently, a 16-mer peptide termed Tip (Transcription inducing peptide; Figure 1A) was isolated via phage display. Tip is not only able to bind specifically to its target, the bacterial transcription factor Tet repressor (TetR), but also triggers a conformational change in vivo normally induced by tetracyclines, the natural effectors of TetR (10). TetR is widely used to control regulation of gene expression in prokaryotes and, as a fusion protein with an activation domain, in eukaryotes (11), because it combines high specificity for its cognate DNA sequence (tetO) with extremely sensitive induction by tetracyclines (tc), especially the potent analogues doxycycline (dox) and anhydrotetracycline (atc) (12,13). Non-tc inducers of TetR, like Tip, may lead to alternative inducers when used as a scaffold for peptidomimetics to generate novel, small-molecule effectors. The recently published crystal structure of the TetR–Tip complex provides a molecular basis for such efforts (14). Another application of Tip is to fuse it to a protein of interest and use it to analyse target protein expression in vivo by monitoring a TetR-controlled reporter (15). Although a wide variety of tag-systems suitable for diverse applications exist, Tip is the only protein tag that allows quantitative in vivo analysis of protein expression. Mandatory for this application is a high efficiency of induction to ensure that low levels of Tip fusion proteins can be detected. We demonstrate here that addition of an aromatic residue between C-terminally fused Tip and TrxA along with mutations in the effector-binding pocket of TetR lead to a strong increase of reporter induction. Furthermore, the introduced TetR mutations render it insensitive to induction by tetracyclines, thereby creating exclusive specificity for Tip.Figure 1.

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