STEPWISE SELECTION OF TETR VARIANTS RECOGNIZING TET OPERATOR 4C WITH HIGH-AFFINITY AND SPECIFICITY

The TetR PQ39 mutant exhibits a new recognition specificity for the te tO-4C operator, but the affinity is not sufficiently high for use in v ivo. A stepwise selection of additional mutations by cassette mutagene sis with randomization of residues in the TetR alpha-helix-turn-alpha- helix motif (HTH) yielded mutant TetR EA37PQ39YM42 showing a similar a ffinity and increased specificity for tetO-4C as wild-type TetR for te tO. A set of mutants obtained by that approach revealed that the fourt h residue of the HTH (Leu41), which points towards the core of the DNA binding domain in TetR, alters the recognition of base-pair 4, e.g. t he mutant TetR LV41YM42 exhibits anew recognition specificity for tetO -4G. A small residue at the last position in the turn of the HTH incre ases the affinity and specificity of DNA binding of TetR mutants conta ining the PQ39 exchange. Thus, cooperation between residues at positio ns 37, 39, 41 and 42 in the HTH of TetR is necessary to optimize recog nition of base-pair 4. We conclude that creating a new DNA recognition specificity in the HTH of TetR with high affinity for the tetO-4C ope rator variant requires exchanges altering flexibility and/or adjustmen t of the recognition a-helix to the target DNA in addition to the cont acting residue. (C) 1998 Academic Press Limited.