A BACTERIAL 2-HYBRID SYSTEM BASED ON A RECONSTITUTED SIGNAL-TRANSDUCTION PATHWAY

We describe a bacterial two-hybrid system that allows an easy in who s creening and selection of functional interactions between two proteins , This genetic test is based on the reconstitution, in an Escherichia coil cya strain, of a signal transduction pathway that takes advantage of the positive control exerted by cAMP. Two putative interacting pro teins are genetically fused to two complementary fragments, T25 and T1 8, that constitute the catalytic domain of Bordetella pertussis adenyl ate cyclase. Association of the two-hybrid proteins results in functio nal complementation between T25 and T18 fragments and leads to cAMP sy nthesis. Cyclic AMP then triggers transcriptional activation of catabo lic operons, such as lactose or maltose, that yield a characteristic p henotype. Hla this genetic test, time involvement of a signaling casca de offers the unique property that association between the hybrid prot eins can be spatially separated from the transcriptional activation re adout. This permits a versatile design of screening procedures either for ligands that bind to a given ''bait,'' as in the classical yeast t wo-hybrid system, or for molecules or mutations that block a given int eraction between two proteins of interest.