AraC protein can activate transcription from only one position and when pointed in only one direction

At the araBAD promoter, the RNA polymerase-proximal half-site for AraC binding partially overlaps the -35 region. Random and explicit spacing experiments show that both this partial overlapping and AraC binding to the polymerase-proximal half-site are necessary and sufficient for strong transcriptional activation. Normally, this occupancy is generated by the presence of arabinose, which shifts AraC from a DNA looping interaction involving the polymerase-distal half-site and the araO2 site 210 base-pairs away, to an interaction with the two half-sites adjacent to RNA polymerase. Changing the polymerase-proximal half-site to a higher affinity AraC binding site gives activation in the absence of arabinose. Thus, arabinose is not required to transform AraC into an activating conformation. Because the two half-sites of araI are direct repeats, the RNA polymerase proximal and distal surfaces of AraC are not identical. When the araI site was turned around, no spacings were found from which AraC could activate transcription. In light of the strict spacing and orientation requirements for AraC activation, the interactions between AraC and RNA polymerase are likely to be specific and inflexible.