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. The two amino acid exchanges in TetR E37A P39K are sufficient for tetO-4C5G specific binding. The E37A mutation increases the affinity of TetR for tetO variants and seems to be essential for binding to modified operator sequences. The Lys39 residue is in a position to directly contact the fourth and fifth bps of tetO thereby creating specificity for tetO-4C5G. Combinations of these mutations with others that lead to a reverse phenotype or altered inducer specificity yielded new TetR mutants with the respective combined activities. Single chain TetR variants were constructed that contain DNA reading heads with two different operator binding specificities. Specific binding of this TetR mutant to the respective mixed tetO-wt/4C5G variants containing one wild type and one double exchange operator half site was only accomplished at a low expression level of TetR variant, while cross-talk with other operator variants were observed at an elevated expression level. This observation emphasizes the importance of the transcription factor expression level for in vivo DNA binding specificity. These new TetR variants can be useful for multigene regulation systems.