title: The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo.
abstract: The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors. However, none of the four mutant repressors binds DNA in a corepressor-independent manner. Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro. Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins. These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo.
authors.affiliation: Department of Biological Sciences, University of Southern California, Los Angeles 90089.
authors.forename: M
authors.initials: M
authors.lastname: Shapiro
authors.forename: D N
authors.initials: DN
authors.lastname: Arvidson
authors.forename: J
authors.initials: J
authors.lastname: Pfau
authors.forename: P
authors.initials: P
authors.lastname: Youderian
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins