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Two-hybrid screening
Molecular biology technique / From Wikipedia, the free encyclopedia
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Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs)[1] and protein–DNA interactions[2][3] by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
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A. The Gal4 transcription factor gene produces a two-domain protein (BD and AD) essential for transcription of the reporter gene (LacZ).
B,C. Two fusion proteins are prepared: Gal4BD+Bait and Gal4AD+Prey. Neither of them are usually sufficient to initiate transcription (of the reporter gene) alone.
D. When both fusion proteins are produced and the Bait part of the first fusion protein interacts with the Prey part of the second, transcription of the reporter gene occurs.
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 two-hybrid screening, the transcription factor is split into two separate fragments, called the DNA-binding domain (DBD or often also abbreviated as BD) and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription.[1][2] The Y2H is thus a protein-fragment complementation assay.