Finding a Mate

Available Two-hybrid Systems Graphic: Leza BerardoneGenome sequencing has produced a vast supply of proteins in need of a functional identity. One way to identify a protein's function is to identify its interacting partners, because proteins often work in pairs or as part of large complexes. Scientists traditionally have used biophysical or biochemical methods (such as affinity chromatography or co-immunoprecipitation) to study protein-protein interactions. More recently, two-hybrid and phage-d

Written byBarbara Cunningham

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The two-hybrid system is an in vivo assay, so proteins are more likely to be in their native conformation, and weak and/or transient interactions that may evade detection in vitro can be detected. Conventional two-hybrid systems are based on the fact that many eukaryotic transcriptional activators contain two physically and functionally separate domains, both of which are required for activity. The DNA-binding domain (DBD) binds to specific cis-regulatory sequences, whereas the activation domain (AD) recruits and instructs RNA polymerase II to transcribe downstream genes. While the DBD and AD are generally part of the same protein, as in the native yeast GAL4 protein, they may also function separately provided they are linked in some way that tethers the AD to the promoter.

The standard yeast two-hybrid system identifies an interaction between two proteins by reconstituting active GAL4 protein.1 The two proteins are expressed as fusions with the GAL4 DBD and ...

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