Mutant Trp repressors with new DNA-binding specificities.
Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of the Escherichia coli trpR (tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes. Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensus trp operator. Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities. These new specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair. The Trp repressor does not use the first two amino acids of its "recognition alpha-helix," Ile79 and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites.
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005973:Glutamine / Q000235:genetics; D007532:Isoleucine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D009154:Mutation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013912:Threonine / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D001059:Apoproteins; D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D014157:Transcription Factors; D007532:Isoleucine; D005973:Glutamine; D013912:Threonine; D008239:Lysine; D000409:Alanine