Probing met repressor-operator recognition in solution.
The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. 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 beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-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 complex, 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 beta-strand motif and that dimer-dimer interactions are required for effective repression.
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Department of Genetics, University of Leeds, UK.
McNally
T
T
Manfield
I
I
Navratil
O
O
Old
I G
IG
Phillips
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SE
Saint-Girons
I
I
Stockley
P G
PG
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D005786:Gene Expression Regulation; D005809:Genes, Regulator / Q000502:physiology*; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008715:Methionine / Q000235:genetics*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D001616:beta-Galactosidase / Q000096:biosynthesis
D011189:Potassium Chloride; D004247:DNA; D008715:Methionine; D001616:beta-Galactosidase