Yeast Two Hybrid Assay: A Fishing Tale

Galactose metabolism Galactose is imported into the cell and converted to galactose-6-phosphate by six enzymes (GAL1, GAL2, PGM2, GAL7, GAL10, MEL1) which are transcriptionally regulated by the proteins Gal80, Gal3, and Gal4, the latter of which plays the central role of DNA-binding transactivator (Figure 1). Gal80 binds Gal4 and inhibits its transcriptional ability. Gal3, in the presence of galactose, binds and causes a conformational change in Gal80, which then allows Gal4 to function as a transcriptional activator . Gal4, like other transcriptional activators, is a modular protein that requires both DNA-binding (BD) and activation domains (AD) . The "two hybrid" technique exploits the fact that Gal4 cannot function as a transcriptional activator unless physically bound to an activation domain. Furthermore, it has been demonstrated that this interaction does not need to be covalent: an experiment was performed where the negative regulatory protein, Gal80, was fused with an activation domain to produce Gal80-AD, and was able to act as a transcriptional activator through its natural binding interaction with a Gal4 protein that was missing its own activation domain . Based on the above observations, a two hybrid assay is performed by expressing two fusion proteins in yeast, the "hunter" and the "bait", where the "hunter" protein is the potential binding partner fused to a yeast activation domain, and the bait is your protein of interest is fused to a yeast binding domain. The hunter and bait constructs are co-transfected into the yeast strain, containing the appropriate “Upstream Activating Sequence” (UAS) proximal to a reporter gene, which is expressed if a binding interaction occurs between the hunter and the bait (Figure 3). To identify proteins that may interact with the bait, a plasmid library, expressing cDNA-encoded AD-fusion proteins, can be screened by introduction into a yeast