Two-hybrid screening
From Freepedia
Two-hybrid screening is a molecular biology technique used to discover protein-protein interactions by testing for physical interactions (such as binding) between two proteins. One protein is termed the bait and the other is a library protein (or prey).
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For the purposes of two-hybrid screening, the transcription factor is split into two separate fragments, called Binding Domain (BD) and Activating Domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for activation of transcription.
The key to the two-hybrid screen is that in most eukaryotic transcription factors, the activating and binding domains are modular and can function in close proximity to each other without direct binding. This means that even though the transcription factor is split into two fragments, it can still activate transcription even if the two fragments are indirectly connected.
The BD fragment is fused onto the bait protein and the AD fragment onto a library protein. If the bait and library interact (i.e. bind), then the AD and BD of the transcription factor would be indirectly connected and transcription of the reporter gene(s) could occur (i.e. transcription of an enzyme to produce tryptophan). If the two proteins do not interact, there would be no transcription of the reporter gene (i.e. the cell would die in tryptophan negative media). Many library proteins can be used to test for interaction with the bait.
Related techniques include one-hybrid screening and three-hybrid screening. The former tests directly for interaction between the library protein and a DNA target and the latter includes a third protein that bridges the bait and library proteins.
A common transcription factor used for yeast two-hybrid screening is GAL4.
Two-hybrid system is similar technique that directly tests for protein-protein interaction where one protein is used as the bait and the other is used as the library protein. Unlike screening, whose purpose is to discover new protein-protein interactions involving multiple library proteins, the two-hybrid system is a test for proposed protein-protein interactions.
