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1.
Two tetracycline repressor (TetR) sequence variants sharing 63% identical amino acids were investigated in terms of their recognition specificity for tetracycline and anhydrotetracycline. Thermodynamic complex stabilities determined by urea-dependent unfolding reveal that tetracycline stabilizes both variants to a similar extent but that anhydrotetracycline discriminates between them significantly. Isofunctional TetR hybrid proteins of these sequence variants were constructed and their denaturation profiles identified residues 57 and 61 as the complex stability determinant. Association kinetics reveal different recognition of these TetR variants by anhydrotetracycline, but the binding constants indicate similar stabilization. The identified residues connect to an internal water network, which suggests that the discrepancy in the observed thermodynamics may be caused by an entropy effect. Exchange of these interacting residues between the two TetR variants appears to influence the flexibility of this water organization, demonstrating the importance of buried, structural water molecules for ligand recognition and protein function. Therefore, this structural module seems to be a key requisite for the plasticity of the multiple ligand binding protein TetR.  相似文献   

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Inducible gene expression based upon Tet repressor (tet regulation) is a broadly applied tool in molecular genetics. In its original environment, Tet repressor (TetR) negatively controls tetracycline (tc) resistance in bacteria. In the presence of tc, TetR is induced and detaches from its cognate DNA sequence tetO, so that a tc antiporter protein is expressed. In this article, we provide a comprehensive overview about tet regulation in bacteria and illustrate the parameters of different regulatory architectures. While some of these set‐ups rely on natural tet‐control regions like those found on transposon Tn10, highly efficient variations of this system have recently been adapted to different Gram‐negative and Gram‐positive bacteria. Novel tet‐controllable artificial or hybrid promoters were employed for target gene expression. They are controlled by regulators expressed at different levels either in a constitutive or in an autoregulated manner. The resulting tet systems have been used for various purposes. We discuss integrative elements vested with tc‐sensitive promoters, as well as tet regulation in Gram‐negative and Gram‐positive bacteria for analytical purposes and for protein overproduction. Also the use of TetR as an in vivo biosensor for tetracyclines or as a regulatory device in synthetic biology constructs is outlined. Technical specifications underlying different regulatory set‐ups are highlighted, and finally recent developments concerning variations of TetR are presented, which may expand the use of prokaryotic tet systems in the future.  相似文献   

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We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.  相似文献   

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The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria–host interface. These studies demonstrate the effectiveness of the tetRtetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.  相似文献   

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To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetR(i2) it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.  相似文献   

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D Porschke  K Tovar  J Antosiewicz 《Biochemistry》1988,27(13):4674-4679
The Tet repressor protein and tet operator DNA fragments and their complexes have been analyzed by electrooptical procedures. The protein shows a positive linear dichroism at 280 nm, a negative linear dichroism at 248 nm, and a strong permanent dipole moment of 3.5 X 10(-27) C m, which is independent of the salt concentration within experimental accuracy. Its rotation time constant of 40 ns indicates an elongated structure, which is consistent with a prolate ellipsoid of 100 A for the long axis and 40 A for the short axis. The time constant can also be fitted by a cylinder of length 78 A and diameter 37 A, which is consistent with nuclease protection data reported on repressor-operator complexes, if the cylinder axis is aligned parallel to the DNA axis. Addition of tetracycline induces changes of the limit dichroism but very little change of the rotation time constant. The rotation time constants observed for the operator DNA fragments show some deviations from the values expected from their contour length; however, these deviations remain relatively small. Formation of repressor-operator complexes leads to some increase of the DNA rotation time constants. Simulations by bead models demonstrate that these time constants can be explained without any major change of the hydrodynamic dimension of the components. The data for the complexes are fitted by bead models with smooth bending of the DNA corresponding to a radius of curvature of 500 A, but at the given accuracy, we cannot rule out that the DNA in the complex remains straight or is bent to a smaller radius of approximately 400 A. Thus, binding of the Tet repressor, which is a helix-turn-helix protein as judged from its sequence, to its operator seems to induce minor bending but does not induce strong bending of the DNA double helix.  相似文献   

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We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant KA of revTetR for binding of [atcMg]+ is ~108 M–1, four orders of magnitude lower than that of TetR. The affinity of TetR for tetO is 5.6 ± 2 × 109 M–1 and that for revTetR in the presence of atc is 1 ± 0.2 × 108 M–1. Both induced forms, the atc-bound TetR and the free revTetR, have the same low affinity of 4 ± 1 × 105 M–1 for DNA. Therefore, atc does not act as a dimerization agent for revTetR. We discuss the structural differences between TetR and revTetR potentially underlying this reversal of activity.  相似文献   

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Controlling gene expression in transgenics   总被引:4,自引:0,他引:4  
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We constructed and characterized four Tet repressor (TetR) variants with engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by tetracycline (tc) might occur. All TetR mutants show nearly wild-type activities in vivo, and the reduced proteins also show wild-type activities in vitro. Complete and reversible disulfide bond formation was achieved in vitro for all four mutants. The disulfide bond in NC18RC94 immobilizes the DNA reading head with respect to the protein core and prevents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thus, these residues must have different conformations when bound to these ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobilize the variable loop between alpha-helices 8 and 9 located near the tc-binding pocket. A faster rate of disulfide formation in the operator-bound conformation and a lack of induction after disulfide formation show that the variable loop is located closer to the protein core in the operator-bound conformation and that a movement is necessary for induction. The disulfide bond in RC195VC199' connects alpha-helices 10 and 10' of the two subunits in the dimer and is only formed in the tc-bound conformation. The oxidized protein shows reduced operator binding. Thus, this bond prevents formation of the operator-bound conformation. The detection of conformational changes in three different regions is the first biochemical evidence for induction-associated global internal movements in TetR.  相似文献   

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The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.  相似文献   

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We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.  相似文献   

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Controlling gene expression in response to stress   总被引:2,自引:0,他引:2  
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