Probing met represser–operator recognition in solution

Abstract

THE three-dimensional crystal structure of the Escherichia coli methionine represser, MetJ, complexed with a DNA operator fragment is described in an accompanying article¹. The complex exhibits several novel features of DNA–protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a β-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized β-strands and DNA², and mutagenesis of the Salmonella phage P22 repressers Arc and Mnt³. The complex comprises a pair of MetJ represser dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel α-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein, and DNA in a previous model of the complex⁴, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the β-strand motif and that dimer–dimer interactions are required for effective repression.