Images on this page were used in our review in Biotechniques

N of citations	Number of citations in Medline that describe use of the yeast two-hybrid system in 1989-1999 is growing exponentially. Total number of citations to date approaches 3000.
Year

	Basic schematic of two-hybrid system to detect interactions between two proteins. As shown, a DNA-binding domain (DBD) fused Bait protein of interest interacts with an Activation-domain (AD)-fused partner protein (prey), either known or selected from a cDNA library. The interacting pair binds a specific sequence motif, activating transcription of at least two separate reporter genes.
The yeast two-hybrid system.
	In this strategy, the DNA-binding domain is fused to a defined protein with the capacity to bind either RNA or a chemical ligand. This fusion construct, termed "Hook" is either coexpressed with a hybrid RNA (Bait) containing the RNA binding site for the Hook and a novel probe sequence; or alternatively, yeast containing the Hook are grown in the presence of a chemically synthesized molecule (Bait) that fuses the Hook ligand and the actual probe sequence. The Prey constitutes an activation-domain fused protein as in other two-hybrid manifestations; in this case, the protein has the property of binding the probe RNA or chemical ligand.
A yeast tribrid system to characterize protein-RNA interactions.
	These reagents allow the discrimination of the interaction of a single prey with two different baits. A first DNA-binding domain fusion (DBD1-B) directs the expression of first set of reporters. A second separate DNA-binding domain fused to a distinct bait (DBD2-C) directs expression of the second set of reporters. This reagent set can be used to select preys that interact with the DBD1-B but not DBD2-C from a library. Alternatively, if starting with a prey that interacts with both DBD1-B and DBD2-C, it can be used to select for mutations or molecules that selectively disrupt the interaction with one of the two baits. See our Dual Bait page for more details.
Dual Bait.
A             B	In the reverse two-hybrid system developed by Vidal et al., reporter 1 (HIS3) is used for positive selection, while reporter 2 (URA3) is used for counterselection.   Top, (A): interaction between DBD- and AD-fused proteins results in growth on medium lacking histidine, but lethality on medium containing 5-fluoroorotic acid (5FOA), a toxic metabolite of the URA pathway.   Below, (B): following mutagenesis of DBD- or AD- fusion protein, missense mutations that weaken the interaction of DBD- and AD-fusions can be separated from nonsense mutants that result in loss of either fusion by comparing profile on histidine- and 5FOA medium: mutations which weaken the interaction will display slow growth on both media, while mutations or truncations which completely abrogate the interaction will result in moderate to strong growth on 5FOA medium, but no growth on histidine- medium.
The reverse two-hybrid system.

For other versions of  Yeast Two-Hybrid systems, see our links