Phenotypes of Combined Tet Repressor Mutants for Effector and Operator Recognition and Allostery

Bertram, Ralph; Kraft, Christine; Wisshak, Stefanie; Mueller, Judith; Scholz, Oliver; Hillen, Wolfgang

doi:10.1159/000084565

Cited by 10 publications

(9 citation statements)

References 39 publications

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“…TetR i2 ‐V99G is one of the best revTetR i2 variants with 4‐ddma‐atc specific repression, almost completely lacking repression with atc [9]. As the V99G exchange in the wild‐type sequence background displays no reverse phenotype and slightly increased repression in the presence of 4‐ddma‐atc, we decided to determine the contribution of each amino‐acid exchange in TetR i2 ‐V99G to the combined activity.…”

Section: Resultsmentioning

confidence: 99%

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Tet repressor mutants with altered effector binding and allostery

et al. 2005

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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 TetRi2 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 TetRi2 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.

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Section: Resultsmentioning

confidence: 99%

“…The combination of mutations resulting in 4‐ddma‐atc specificity with those yielding revTetR in the same polypeptide did not yield efficient mutants with combined activities [9]. Thus, these properties of TetR must be interrelated.…”

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confidence: 99%

Tet repressor mutants with altered effector binding and allostery

et al. 2005

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Section: Modifications Of Tet Regulation: Revtetr and Tetr Mutants Emmentioning

confidence: 99%

“…It was recently demonstrated that the different phenotypic alterations of shifted operator and inducer preference and reverse behaviour of TetR variants can functionally be combined in novel regulators, which, e.g. only in the presence of 4-de-dimethylamino-atc bind to tetO (Bertram et al, 2004;Henssler et al, 2005). A dodecapeptide, termed Tip (transcription-inducing peptide), isolated by phage display has pronounced inducing capacities for TetR(B) (Klotzsche et al, 2005).…”

Section: Modifications Of Tet Regulation: Revtetr and Tetr Mutants Emmentioning

confidence: 99%

The application of Tet repressor in prokaryotic gene regulation and expression

Bertram

Hillen

2007

Microbial Biotechnology

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SummaryInducible 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 tetsystems 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.

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“…Traditional small-molecule-inducible expression systems, such as those derived from the lactose (lac), arabinose (ara) and tetracycline (tet) operons, have been extensively utilized for the production of recombinant proteins, [1] gene function analysis, [2] target validation, and drug discovery. [3] Inducible expression systems have also become indispensable tools in synthetic biology and have been used to control genetic circuits and networks, [4] as well as metabolic pathways for the production of pharmaceuticals, biofuels, and fine chemicals.…”

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confidence: 99%