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 of the helix-turnhelix (HTH) motif of the Trp repressor, and guanine in position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical alpha- or beta-centred trp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interactions with other bases in positions 8 and 9 of the alpha-centred trp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus 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 trp operator shows a change in the specificity of binding to a beta-centred symmetrical trp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the alpha-centred trp operator.
Günes
C
C
Institut für Genetik, Universität zu Köln, Germany.
Müller-Hill
B
B
D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013941:Thymine / Q000378:metabolism; D014364:Tryptophan; D029968:Escherichia coli Proteins
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D013941:Thymine; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins