AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli.

Abstract

We examined the recognition of the araBAD promoter by the AraC protein in the Escherichia coli arabinose operon. A mutant promoter, with base substitutions at positions contacted by AraC, was used to isolate suppressor mutations in araC by direct selection. Two hydroxylamine-induced araC mutations were isolated repeatedly; each contained a single amino acid substitution. When tested against a set of base substitution promoter mutants, one revertant, an Arg to His substitution at residue 250, displayed altered base specificity for a single position within the araBAD promoter. The other revertant, a Cys to Tyr substitution at residue 204, did not show consistent base-specific suppression. Neither demonstrated a higher affinity than the wild type protein for the mutant promoter in vitro. Both proteins suppress mutant sequences by a mechanism that does not appear to involve the formation of new net favorable contacts with the mutant base pairs of the promoter.