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 recogni tion alpha-helix leads to a new DNA recognition specificity for tetO-6 %, however, it is bound with only low affinity. Specificity and affini ty of this mutant were substantially increased by additional amino aci d exchanges in the last positions of the recognition alpha-helix and t he turn, which most likely play structural roles in the formation of t he 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 var iants and contributes efficiently to the affinity for the newly recogn ized tetO-6C sequence. Short residues at this position improve sequenc e specific binding when combined with a residue in the recognition alp ha-helix, which directly reads out the recognized tetO sequence. We as sume that small residues at the end of the turn permit the recognition alpha-helix to assume the optimal position within the motif for clock ing to the DNA target. Thus, residues allowing direct and favourable c ontacts to the newly recognized DNA are not sufficient to increase the binding specificity and affinity, but need to be accompanied by addit ional exchanges allowing the formation of these contacts. (C) 1998 Aca demic Press Limited.