Stepwise selection of TetR variants recognizing tet operator 6C with high affinity and specificity.

The exchange of Trp43 to Arg in the sixth position of the TetR recognition alpha-helix leads to a new DNA recognition specificity for tetO-6C, however, it is bound with only low affinity. Specificity and affinity of this mutant were substantially increased by additional amino acid exchanges in the last positions of the recognition alpha-helix and the turn, which most likely play structural roles in the formation of the TetR-tetO complex. The last residue in the turn of the alpha-helix-turn-alpha-helix motif is a discriminator of binding to other tetO variants and contributes efficiently to the affinity for the newly recognized tetO-6C sequence. Short residues at this position improve sequence specific binding when combined with a residue in the recognition alpha-helix, which directly reads out the recognized tetO sequence. We assume that small residues at the end of the turn permit the recognition alpha-helix to assume the optimal position within the motif for docking to the DNA target. Thus, residues allowing direct and favourable contacts to the newly recognized DNA are not sufficient to increase the binding specificity and affinity, but need to be accompanied by additional exchanges allowing the formation of these contacts.