Two Hybrid Screen

---

---

Two-hybrid screening (also known as yeast two-hybrid system or Y2H) is a technique used to test if two proteins interact with each other. The assay makes use of a transcription factor that has been "cut in half" to separate the binding domain from the activating domain. Typically, the binding domain of the transcription factor would attach to an upstream activation sequence on DNA, bringing the activating domain in close proximity to the promoter, allowing it to interact with the transcription machinery. We call the gene being transcribed the reporter gene because if it's actively being transcribed, it leads to the expression of a measurable protein. However, because the binding domain and the activating domain are separated in this type of assay, no transcription takes place. Unless...

Unless somehow the two domains could be joined back together. Now consider how this sets the stage for detecting protein-protein interactions. By creating two hybrids, one containing a binding domain fused to a protein of interest (binding domain + protein¹ = bait), the other with the activating domain fused to another protein (activating domain + protein² = prey).

The bait hybrid can bind to the DNA, but cannot activate transcription. The prey hybrid can activate transcription but has little chance to come in close proximity to the appropriate gene segment. However, if protein¹ and protein² interact with each other, then transcription can ensue. And we know when transcription ensues if the cell is manufacturing the protein that the reporter gene codes. The technique has been combined with a number of different reporter genes which can allow selection through a simple colour change or through automatic death of cells in which the interaction does or does not take place. For example, the lacZ reporter gene allows the highlighting of cells in which, β-galactosidase, the protein product of the lacZ gene produces a blue coloration through the metabolism of X-gal.

---