Engineered Tet repressors with recognition specificity for the tetO-4C5G operator variant.

We created a new DNA recognition specificity for tetracycline repressor (TetR) binding to the tet operator variant tetO-4C5G containing four bp exchanges compared to tetO. TetR variants created by doped oligonucleotide mutagenesis of residues in the DNA recognition helix yielded several mutants binding to tetO-4C5G. These variants contained exchanges of the amino acids at positions 36, 37, 39 and 42.

Tet repressor mutants with altered effector binding and allostery.

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.

Phenotypes of combined tet repressor mutants for effector and operator recognition and allostery.

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.

Activity reversal of Tet repressor caused by single amino acid exchanges.

We 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.