Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP

Abstract

Expression from the E.coli melR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP. We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences. When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP. Single changes at either of two symmetrically-related positions create sites that are recognised by both FNR and CRP. Changes at both positions result in a site that is not recognised by FNR but which binds CRP tightly.