Figure 1. Reverse bacterial two hybrid system.

A: In the E. coli two-hybrid interacting proteins X and Y fused to B. pertussis adenylate cyclease T18 and T25 fragments will restore enzyme activity, resulting in cAMP production, and in association with the catabolite activator protein (CAP) activation of cAMP dependent promoters In the reverse two-hybrid system, the pyrF gene was placed downstream of the lac promoter, hence activation by cAMP results in pyrF expression. The pyrF gene encodes orotidine-5 ′-phosphate decarboxylase which can convert 5-fluoroorotic acid (5-FOA) to the toxic compound 5-flourouracil whuich results in inviability. If the interaction between X and Y is abolished by a small compound (filled red circle) cAMP will no longer be produced and viability restored. B: Fusion of DnaN to both T18 and T25 results in inhibition of growth on 5-FOA plates due to reconstitution of a functional Cya protein. In the absence of fusion partners T18 and T25 will not form a functional andenylate cyclase. Therefore pyrF is not expressed and cells remain viable in the presence of 5-FOA.