In vitro evolution of the DNA binding sites of Escherichia coli methionine repressor, MetJ

The SELEX procedure was used to study the recognition between the E. coli methionine repressor (MetJ) and its DNA binding sites. DNA ligands with high affinity for either the hole-repressor or ape-repressor were isolated from a pool of molecules randomized over 20 base-pairs. Among 90 DNA ligands selected by hole-repressor binding, roughly 90% contain variations of two tandem, perfect eight base-pair Met-boxes, which are the consensus deduced from natural met operators. Base-pairs that are important, for specific interactions with the protein are highly conserved. The data also reveal the importance of the non-contacted operator base-pairs in facilitating the conformational changes in the operator which must occur for repressor binding. There are also effects due to the sequences of the base-pairs immediately flanking the operator site. DNA ligands selected by apo-repressor share a very similar, but not identical, consensus with that selected by hole-repressor, suggesting that the corepressor does not greatly alter the specificity of repressor binding. (C) 1996 Academic Press Limited