Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription

Abstract

The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures.