An Exclusive α/β Code Directs Allostery in TetR–Peptide Complexes

Sevvana, Madhumati; Goetz, C; Goeke, Dagmar; Wimmer, Cornelius; Berens, Christian; Hillen, Wolfgang; Muller, Yves A.

doi:10.1016/j.jmb.2011.12.008

Cited by 18 publications

(15 citation statements)

References 47 publications

(69 reference statements)

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“…In contrast, M10 and W11 might be important by forming strong interactions with rtTA via their functional groups. A similar result was found for the TetR inducing peptides TIP and TIP2, since the crystal structures of both, [TetR⋅TIP] and [TetR⋅TIP2], revealed tryptophan residues localized within the tc-binding pocket forming crucial interactions to residues of TetR [28], [30]. For both peptides, the activity was lost when the tryptophan residues were exchanged to alanine [29], [31].…”

Section: Resultssupporting

confidence: 76%

“…A similar behavior was observed with TetR-regulating peptides TIP2 and TAP1, whose N-terminal residues are permissive to alanine exchanges, while their C-terminal residues are not [29]. Since the crystal structures of [TetR⋅TIP], [TetR⋅TIP2] and [TetR⋅TAP1] complexes revealed at least partial binding of the peptide inside the tc-binding pocket [28], [30], a feasible conclusion is that binding of K79 residues 9 to 16 also occurs inside the rtTA effector binding pocket.…”

Section: Resultssupporting

confidence: 60%

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A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

2014

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Conditional regulation of gene expression is a powerful and indispensable method for analyzing gene function. The “Tet-On” system is a tool widely used for that purpose. Here, the transregulator rtTA mediates expression of a gene of interest after addition of the small molecule effector doxycycline. Although very effective in rapidly turning on gene expression, the system is hampered by the long half-life of doxycycline which makes shutting down gene expression rapidly very difficult to achieve. We isolated an rtTA-binding peptide by in vivo selection that acts as a doxycycline antagonist and leads to rapid and efficient shut down of rtTA-mediated reporter gene expression in a human cell line. This peptide represents the basis for novel effector molecules which complement the “Tet-system” by enabling the investigator to rapidly turn gene expression not just on at will, but now also off.

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Section: Resultssupporting

confidence: 76%

Section: Resultssupporting

confidence: 60%

A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

2014

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“…Indeed, it has been shown previously that highly artificial ligands such as peptides are able to efficiently induce TetR [21]. Although such inducers do not mimic the exact binding mode of antibiotics, their presence in the binding site elicits the same conformational effects at the level of the a6-a7 segment [22]. We believe that the highly adaptable, modular repressor design found in TetR has been a key factor in the evolutionary success of the repressor family, and note that comparable modular mechanisms are likely to have emerged in other wide-spread classes of ligand-binding transcription factors.…”

Section: Discussionmentioning

confidence: 99%

Modular organisation of inducer recognition and allostery in the tetracycline repressor

et al. 2016

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Induction of the tetracycline repressor (TetR) results from antibioticdependent changes in the relative positioning of the DNA-binding domains within the promoter-associated repressor dimer, but the key determinants of this allosteric effect remain poorly characterised. Intriguingly, previous mutational analyses of the tetracycline-interacting site revealed a lack of correlation between residual affinity and induction propensity, suggesting that some of the residues in contact with the antibiotic primarily act in ligand recognition and retention, whereas others are required to transmit the allosteric signal. Here, we provide a structural basis for these observations via crystallographic analysis of the point mutants N82A, H100A, T103A and E147A in complex with the inducer 5a,6-anhydrotetracycline. In conjunction with the available functional data, the four structures demonstrate that a trigger-like movement of the region between helices a6 and a7 towards and into the binding site plays a decisive role in the intramolecular communication process. In sharp contrast, residues lining the binding cavity proper have little or no influence on the allosteric mechanism as such. This nearly complete physical separation of ligand recognition and allostery will have allowed diverging TetR-like repressors to bind novel effectors while the existing induction mechanism remained intact. Consequently, the modularity described here may have been a key factor in the evolutionary success of the widespread and highly diversified repressor class.

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“…The recent discovery that peptides can also specifically induce TetR when they are fused to a carrier protein [19]–[21] added a new quality to Tet regulation. These inducing peptides, called TIP (TetR-inducing peptide), bind to the tetracycline-binding pocket of TetR and elicit an allosteric conformational change that leads to the complete loss of DNA-binding activity [22], [23]. This turned TetR from an exclusively small-molecule-controlled protein into a downstream effector in a protein signal transduction pathway.…”

Section: Introductionmentioning

confidence: 99%

Promoter Strength Driving TetR Determines the Regulatory Properties of Tet-Controlled Expression Systems

et al. 2012

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Bacteria frequently rely on transcription repressors and activators to alter gene expression patterns in response to changes in the surrounding environment. Tet repressor (TetR) is a paradigm transcription factor that senses the environmental state by binding small molecule effectors, the tetracyclines. However, recently isolated peptides that act as inducers of TetR after having been fused to the C-terminus of a carrier protein, suggest that TetR can also regulate gene expression in a signal-transduction pathway. For this shift in regulatory mechanism to be successful, induction of TetR must be sensitive enough to respond to an inducing protein expressed at its endogenous level. To determine this regulatory parameter, a synthetic Tet-regulated system was introduced into the human pathogen Salmonella enterica serovar Typhimurium and tested for inducibility by a peptide. Reporter gene expression was detected if the peptide-containing carrier protein Thioredoxin 1 was strongly overproduced, but not if it was expressed at a level similar to the physiological level of Thioredoxin 1. This was attributed to high steady-state amounts of TetR which was expressed by the promoter of the chloramphenicol acetyl transferase gene (Pcat). Reducing Pcat strength either by directed or by random mutagenesis of its -10 element concomitantly reduced the intracellular amounts of TetR. Sensitive and quantitative induction of TetR by an inducing peptide, when it was fused to Thioredoxin 1 at its native locus in the genome, was only obtained with weak Pcat promoter variants containing GC-rich -10 elements. A second important observation was that reducing the TetR steady-state level did not impair repression. This permits flexible adjustment of an inducible system’s sensitivity simply by altering the expression level of the transcription factor. These two new layers of expression control will improve the quality and, thus, the applicability of the Tet and other regulatory systems.

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An Exclusive α/β Code Directs Allostery in TetR–Peptide Complexes

Cited by 18 publications

References 47 publications

A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

Modular organisation of inducer recognition and allostery in the tetracycline repressor

Promoter Strength Driving TetR Determines the Regulatory Properties of Tet-Controlled Expression Systems

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