AraC protein contacts asymmetric sites in the Escherichia coli araFGH promoter

AraC protein regulates the transcription of arabinose transport and catabolic operons in Escherichia coli through interaction with specific DNA sequences in the promoter regions of the operons. The interaction of AraC protein with two binding sites in the araFGH promoter was determined and compared to previously studied AraC binding sites in the araBAD promoter. Methylation and ethylation interference assays show that AraC protein binds along one side of the DNA to four adjacent major groove regions at each of the araFG1 and araFG2 sites. Mutations within any of the four regions of araFG1 greatly reduce protein binding in vitro. The promoter function in vivo is also greatly reduced, indicating that all four regions of the binding site are required. The chemical interference and genetic data, combined with the consensus sequence for AraC protein binding to ara promoters, support a binding motif in which two directly repeated units each span two adjacent turns of the DNA helix. The function of the two AraC binding sites was also examined. The proximal araFG1 site is required for promoter activation, whereas the distal araFG2 site has only a slight effect on the promoter activity.