Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain.
The NH2-terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor-operator complexes via association with non-operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA. To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions. Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons. Positions 4, 5 and 6 were the most sensitive to missense changes. Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild-type trp repressor. A large percentage of the missense mutants were more active than the wild-type repressor in medium containing tryptophan and less active in medium without tryptophan. This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor. Also, nine super-repressor mutants, resulting from substitutions clustered at both ends of the arms, were found. Our results support the hypothesis that the NH2-terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.
Mackintosh
S G
SG
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
McDermott
P F
PF
Hurlburt
B K
BK
D001426:Bacterial Proteins; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry*; D017930:Genes, Reporter / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics; D009876:Operon / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins