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yeast two-hybrid system


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An in vivo (q.v.) method for identifying protein-protein interactions, based on the properties of a transcriptional activator protein. The simplest version of this system is based on the yeast protein, GAL4 (q.v.), whose DNA-binding domain (BD) binds with an upstream activator sequence (UAS) and the activation domain (AD) interacts with the transcription complex to stimulate transcription of a downstream gene (see illustration A). In this two-hybrid scheme, two plasmids (q.v.) encoding two hybrid proteins are constructed and introduced into yeast cells. One hybrid contains the GAL4 BD fused to a known protein (protein X), and the second hybrid is a fusion between the GAL4 AD and a second protein (protein Y). These hybrids are coexpressed in a yeast strain lacking GAL4 activity and containing a reporter gene (q.v.), such as the bacterial lac Z gene, with a binding site for GAL4. Either hybrid by itself is incapable of inducing transcription (illustration B). An interaction between proteins X and Y, however, brings the BD and AD in close proximity, and GAL4 activity is reconstituted (illustration C). This leads to transcriptional activation of the reporter gene and allows X-Y interaction to be monitored by β-galactosidase (q.v.) activity. This approach has been modified to screen protein sequences in libraries. In this case, the second hybrid is a fusion between the GAL4 AD and proteins encoded by genomic (q.v.) or cDNA library (q.v.) sequences. Interaction between the known protein and a protein encoded by one of the library plasmids is detected by expression of the reporter gene. See Chronology, 1989, Field and Song; GAL4, lac operon.

Illustration of yeast two-hybrid system by Vikram K. Mulligan.

Subjects: Genetics and Genomics — Chemistry.


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