Effects of systematic variation of the minimal Escherichia coli met consensus operator site: In vivo and in vitro met repressor binding

We have produced a set of sequence variants based upon the idealized, minimal Escherichia coli met operator in which each position within the basic recognition unit, the 8 bp met box (dAGACGTCT), has been changed to all other possible sequences containing single symmetrical base substitutions. The effects of these sequence variations have been assayed in vivo by monitoring the production of beta-galactosidase from a standard promoter regulated by the operator variants, and in vitro by gel-retardation assay. The two sets of data are consistent and correlate well with expectations based on the three-dimensional structure of the holorepressor bound to a minimal idealized operator and the results of in vitro evolution experiments. Comparison with two natural operators, metA and metC, suggests that in vivo, with non-consensus operators, the repressor binds to at least four consecutive met boxes.