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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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Location of Tip in the TetR–Tip complex structure. Residues W1 to A5 of Tip and the residues N82 and F86 of TetR are displayed as gray stick models. Ac indicates the acetyl moiety at the N-terminus of Tip present in the complex. The distance (dashed lines) between the Cα of the acetyl and the Cβ atoms of N82 or F86 is indicated. The dotted line indicates the continuation of Tip. The coordinates were taken from the PDB-file 2NS8 (14).
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Figure 2: Location of Tip in the TetR–Tip complex structure. Residues W1 to A5 of Tip and the residues N82 and F86 of TetR are displayed as gray stick models. Ac indicates the acetyl moiety at the N-terminus of Tip present in the complex. The distance (dashed lines) between the Cα of the acetyl and the Cβ atoms of N82 or F86 is indicated. The dotted line indicates the continuation of Tip. The coordinates were taken from the PDB-file 2NS8 (14).

Mentions: X-ray crystallography of TetR complexed with a 16-mer Tip oligopeptide acetylated at the N-terminus revealed details of the location of Tip inside the tc-binding pocket. The Cα atom of the acetyl moiety is in close proximity to residues N82 and F86 of TetR (14). Figure 2 shows an excerpt of the structure highlighting this proximity. We assumed that a large residue at this position, as found in several active C-terminal TrxA(C)-XTip variants, might lead to sterical hindrance. Therefore, we replaced the bulky N82 and F86 residues with a smaller A residue and scored the induction properties of these mutants with TrxA(C)-MTip. The results are shown in Figure 3. Even in the absence of IPTG, in which only basal expression of TrxA(C)-MTip from the leaky tac promoter occurs, we observed induction of TetR-N82A and TetR-F86A. However, TrxA(N)-Tip is still the more efficient inducer under these conditions. At the higher, IPTG-induced, expression level of TrxA(C)-MTip, induction of TetR-N82A reaches the same level as with TrxA(N)-Tip and wt TetR. A slightly smaller maximal induction by TrxA(C)-MTip is observed for TetR-F86A. As expected, altering the size of the binding pocket in TetR-N82A and TetR-F86A does not lead to increased induction by TrxA(C)-Tip lacking the additional M residue.Figure 2.


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)

Location of Tip in the TetR–Tip complex structure. Residues W1 to A5 of Tip and the residues N82 and F86 of TetR are displayed as gray stick models. Ac indicates the acetyl moiety at the N-terminus of Tip present in the complex. The distance (dashed lines) between the Cα of the acetyl and the Cβ atoms of N82 or F86 is indicated. The dotted line indicates the continuation of Tip. The coordinates were taken from the PDB-file 2NS8 (14).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919500&req=5

Figure 2: Location of Tip in the TetR–Tip complex structure. Residues W1 to A5 of Tip and the residues N82 and F86 of TetR are displayed as gray stick models. Ac indicates the acetyl moiety at the N-terminus of Tip present in the complex. The distance (dashed lines) between the Cα of the acetyl and the Cβ atoms of N82 or F86 is indicated. The dotted line indicates the continuation of Tip. The coordinates were taken from the PDB-file 2NS8 (14).
Mentions: X-ray crystallography of TetR complexed with a 16-mer Tip oligopeptide acetylated at the N-terminus revealed details of the location of Tip inside the tc-binding pocket. The Cα atom of the acetyl moiety is in close proximity to residues N82 and F86 of TetR (14). Figure 2 shows an excerpt of the structure highlighting this proximity. We assumed that a large residue at this position, as found in several active C-terminal TrxA(C)-XTip variants, might lead to sterical hindrance. Therefore, we replaced the bulky N82 and F86 residues with a smaller A residue and scored the induction properties of these mutants with TrxA(C)-MTip. The results are shown in Figure 3. Even in the absence of IPTG, in which only basal expression of TrxA(C)-MTip from the leaky tac promoter occurs, we observed induction of TetR-N82A and TetR-F86A. However, TrxA(N)-Tip is still the more efficient inducer under these conditions. At the higher, IPTG-induced, expression level of TrxA(C)-MTip, induction of TetR-N82A reaches the same level as with TrxA(N)-Tip and wt TetR. A slightly smaller maximal induction by TrxA(C)-MTip is observed for TetR-F86A. As expected, altering the size of the binding pocket in TetR-N82A and TetR-F86A does not lead to increased induction by TrxA(C)-Tip lacking the additional M residue.Figure 2.

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