Schematic of the bacterial one-hybrid reporter system. In the new bacterial one-hybrid system, a selected promoter sequence or the other genomic DNA, the bait DNA, was cloned into the reporter vector of pBXcmT containing the selectable genes HIS3 and aadA. A given transcription factor or a library was expressed as a fusion to the alpha subunit of the RNA polymerase in the vector of pTRG. The reporter genes would be activated in case of interaction of the promoter region with a given regulator protein expressed in pTRG when a pair of recombinant vectors is cotransformed into the host strain. If the regulator protein interacts with the target DNA, it recruits and stabilizes the binding of RNA polymerase at the weak promoter and thus activates the transcription of the HIS3 reporter gene in the presence of the compound 3-amino-1,2,4-triazole (3-AT). A second reporter gene aadA encoding a protein that confers streptomycin resistance provides an additional mechanism to validate the DNA–protein interaction.
