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Nucleic Acids Research, 1993, Vol. 21, No. 24 5661-5666
© 1993

MOLECULAR BIOLOGY

The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp superrepressors in vivo

Michael Shapiro+, Dennis N. Arvidson, James Pfau and Philip Youderian*

Department of Biological Sciences University of Southern California, Los Angeles, CA 90089 and California Institute of Biological Research 11099 N. Torrey Pines Rd, La Jolla, CA 92037, USA

*To whom correspondence should be addressed

Received August 31, 1993. Revised November 1, 1993. Accepted November 1, 1993.

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{twoheadrightarrow}Lys, Glu18{twoheadrightarrow}Lys, Glu49{twoheadrightarrow}Lys and Ala77{twoheadrightarrow}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 corepressorindependent manner. Three of the four mutant repressors (with Glu{twoheadrightarrow}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.

+Present address: Department of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, USA


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