Three adjacent binding sites for cAMP receptor protein are involved in the activation of the divergent malEp-malKp promoters

Abstract

The divergent malEFG and malK-lamB-malM operons in Escherichia coli are controlled by partially overlapping promoters, whose activity depends on the presence of two transcriptional activators, MalT and the cAMP receptor protein (CRP). The 271-base-pair region separating the transcription start points of the promoters malEp and malKp comprises a compact array of binding sites for MalT and CRP. We report the characterization of the in vitro interactions of CRP with its four adjacent binding sites and the analysis of their function in vivo. By using the DNase I footprinting technique, we showed that CRP binds with high affinity to the three malEp-proximal sites and with a low affinity to the fourth site. CRP binding to these sites is not cooperative, even though they are adjacent and located on the same face of the DNA double helix. Each of these sites was destroyed by localized mutagenesis and the residual activity of the promoters was measured in vivo. Mutations in any of the three high-affinity binding sites reduced both malEp and malKp activity. The participation of several adjacent bound CRP molecules in the activation of a promoter is an unprecedented observation and might involve molecular mechanisms quite different from those used in the other CRP-controlled promoters.