SummaryWe explore by extensive mutagenesis regions in the sequence allowing reversal of the allosteric response of Tet repressor. The wild type requires anhydrotetracycline for induction. About 100 mutants are presented, which, in contrast, require the drug for repression. Their mutations are clustered at the interface of the DNA-and inducer-binding domains. This interface consists of a central hydrophobic region surrounded by several hydrogen bonds. While most of the mutants described here contain two to five mutations, we found five positions in this region of TetR, at which single amino acid exchanges lead to activity reversal. They may disrupt the hydrogenbonding network bordering the domain interface. We assume that the mutations cause a repositioning of the DNA reading head with respect to the effector binding core so that the same conformational change can result in opposite activities.

No abstract

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.

Tet repressor mutants with a shifted effector specificity, preference for a mutant operator sequence or reversion of activity were combined to construct variants bearing two or three phenotypic alterations. TetR alleles with combinations of altered operator and effector specificities can be created by merging the respective residues in a single polypeptide. The mutations giving rise to revTetR, on the other hand, show drastic influences on the ligand binding phenotypes when combined with respective alterations. One TetR variant displays all three phenotypic alterations and thus demonstrates the general possibility of implementing them in one protein.