Building protein-protein networks by two-hybrid mating strategy

Abstract : The basic concept of the yeast two-hybrid system is to detect the interaction between two proteins via transcriptional activation of a reporter gene. A classical eukaryotic transcription activator contains a domain that specifically binds to DNA sequences and a domain that recruits the transcription machinery. The yeast two-hybrid system can detect interactions between two known proteins or polypeptides and can also search for unknown partners of a given protein. The yeast two-hybrid system is an indirect genetic assay. Intrinsic limitations of the yeast two-hybrid system include reliance on complex transcriptional activation of reporter genes. Incorrect folding, inappropriate subcellular localization (bait and prey proteins must interact in a nuclear environment), or degradation of chimeric proteins and absence of certain types of posttranslational modifications in yeast could lead to false negatives. Other properties of the assay may lead to the selection of false positives. Confirmation of interactions should be obtained via various independent genetic, biochemical or functional assays, such as copurification in complexes, colocalization, or demonstration of a functional association.