MUTANTS IN POSITION-69 OF THE TRP REPRESSOR OF ESCHERICHIA-COLI K12 WITH ALTERED DNA-BINDING SPECIFICITY

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix o f the helix-turn-helix (HTH) motif of the Trp repressor, and guanine i n position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gin an d tested the resultant repressor mutants for their binding to syntheti c symmetrical alpha- or beta-centred tup operator variants, in vivo an d in vitro. We present genetic and biochemical evidence that Ile in po sition 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interacti ons with other bases in positions 8 and 9 of the alpha-centred trp ope rator. In vitro, the Trp repressor of mutant RI69 binds to the consens us alpha-centred trp operator and a similar trp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the beta-centred t rp operator shows a change in the specificity of binding to a beta-cen tred symmetrical trp operator variant with a gua-nine to thymine subst itution in position 5, which corresponds to position 9 of the alpha-ce ntred trp operator.