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 coil met operator in which each position within th e basic recognition unit, the 8 bp met box (dAGACGTCT), has been chang ed to all other possible sequences containing single symmetrical base substitutions. The effects of these sequence variations have been assa yed 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 correla te well with expectations based on the three-dimensional structure of the holorepressor bound to a minimal idealized operator and the result s of in vitro evolution experiments. Comparison with two natural opera tors, metA and metC, suggests that in vivo, with non-consensus operato rs, the repressor binds to at least four consecutive met boxes.