Reverse Y2H system

Fig. 7.4. Variations of the original Y2H assay have been designed to detect the disruption of protein-protein interactions. A. In the reverse Y2H system, a direct protein-protein interaction between X and Y activates the transcription of URA3, which converts 5-FOAto a toxic compound (left) however, if the interaction between X and Y is disrupted, then transcription of URA3 is disrupted and the cells can grow in media containing 5-FOA (right) [28]. B. In the split-hybrid system, a direct protein-protein interaction between X and Y ac-...

As with other in vivo selections, it is critical to validate hits at the end of the selection experiment with a secondary screen. The most common secondary screen used with the Y2H assay is a lacZ screen. If either the DBD or AD fusion is under control of an inducible promoter, this screen can be carried out both under inducing and non-inducing conditions to ensure that transcription activation is protein dependent. Y3H systems, where transcription activation depends on a bridging RNA or small molecule, and reverse Y2H systems, where a third protein is disrupting the interaction, provide a built-in control. One can simply check that transcription activation is in fact dependent on the third component. As with any in vivo selection, the evolved plasmid should be isolated and retransformed into a fresh yeast selection strain to ensure that the phenotype is plasmid-dependent. Ultimately, the interaction will be confirmed with co-immunoprecipitation experiments or other in vitro binding assays [39]. 

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