Enhanced operator binding by trp superrepressors of Escherichia coli.
The trp repressor of Escherichia coli binds to the operators of three operons concerned with tryptophan biosynthesis and regulates their expression. trp superrepressors can repress expression of the trp operon in vivo at lower tryptophan concentrations than those required by the wild-type repressor. The five known superrepressors have been purified and characterized using a modified filter binding assay. In four of the five superrepressors, EK13, EK18, DN46 and EK49, negatively charged wild-type residues located on the surface of the repressor that faces the operator are replaced by positively charged or neutral residues. Each of these proteins has higher affinity for the trp operator than wild-type repressor. Decreased rates of dissociation of the repressor-operator complex were found to be responsible for the higher affinities. The fifth superrepressor, AV77, has an amino acid substitution in the turn of the helix-turn-helix DNA-binding motif. This superrepressor was indistinguishable from wild-type repressor in our filter binding assay. We conclude that rapid dissociation of repressor from operator is important for trp repressor function in vivo. The negatively charged wild-type residues that are replaced in superrepressors are probably responsible for the characteristic rapid dissociation of the trp repressor from the trp operator.
Hurlburt
B K
BK
Department of Biological Sciences, Stanford University, California 94305-5020.
Yanofsky
C
C
D001483:Base Sequence; D001665:Binding Sites; D003101:Collodion; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005374:Filtration; D005971:Glutamates; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D009876:Operon; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014364:Tryptophan / Q000096:biosynthesis* / Q000494:pharmacology
D004269:DNA, Bacterial; D005971:Glutamates; D012097:Repressor Proteins; D014157:Transcription Factors; D018698:Glutamic Acid; D014364:Tryptophan; D003101:Collodion