THE CHALLENGE-PHAGE ASSAY REVEALS DIFFERENCES IN THE BINDING EQUILIBRIA OF MUTANT ESCHERICHIA-COLI TRP SUPER-REPRESSORS IN-VIVO

The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challen ge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes fr om the wild type (Glu13 --> Lys, Glu18 --> Lys, Glu49 --> Lys and Ala7 7 --> Val), require less L-tryptophan than wild-type repressor for act ivation in vivo, and are super-aporepressors underbar. However, none o f the four mutant repressors binds DNA in a corepressor-independent ma nner. Three of the four mutant repressors (with Glu --> Lys changes) a re more active when complexed with tryptophan, and are super-holorepre ssors underbar. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding stud ies in vitro. Challenge-phage assays with limiting tryptophan reveal a dditional phenotypic differences among the mutant proteins. These resu lts show that the challenge-phage assay is a robust assay for measurin g the relative affinities of specific protein - DNA interactions in vi vo.