Yeast two-hybrid screens are an efficient system for mapping protein-protein interactions and whole interactomes. This method also helps researchers identify what other proteins an identified protein can interact with. This ultimately helps form the blueprint of the protein interactome, or the protein network. Transcription activators have two distinct

This protocol focuses on the activation results from a given reporter gene [def A gene used as a selectable marker in terms of observing protein expressions in organisms.]. How it is measured depends on the reporter gene itself. Yeast is the basis for this protocol because it has the ability to grow on nutrient restricted-medium. This protocol definitely has its advantages of having a simple and quick process that results in potentially multiple interactions, as many prey proteins are interacted with a single bait.

The term two-hybrid is derived from the two classes of hybrid protein baits used in each screening. One of the proteins of interest (protein x) is called the bait for the process and the other protein of interest (protein y) is the prey. T he bait is bound to a DNA-binding domain while the prey is bound to a transcriptional activation domain [def Used in order to recognize any activation domains of proteins]. A prey pool was created for the bait strain to determine interactions. This system utilizes the yeast Gal4 activator as the DNA-binding domain to activate the transcription process as well.

Additionally, a LexA-based system was used for a second round of Y2H experiments. This experimental step was included in order to determine any hypothetical proteins [def Existence has been predicted but there is a lack of experimental evidence to solidify the existence.]. This was done by experimentally designing an activation domain array and a binding domain array. The activation domain array consisted of yeast clones and the BD array consisted of the 360 Streptococcus pneumoniae genes. Each BD item (each gene) was then mated with the AD array, resulting in 1,054 interactions being found.

Gal4 is used as the activator, as it has shown to cooperatively bind to activation domains [8 (Vashee & Kodadek, 1995). The activation domain of GAL4 protein mediates cooperative promoter binding with general transcription factors in vivo. Proc Natl Acad Sci USA 1995; 92(23):10683–7.]. Further explained, activation domains frequently bind to the TATA box [def A section of a DNA sequence that locates where a genetic sequence can be coded and read] found in many promoters that initiation transcriptional activity. Ultimately by including this activator, success of determining any interactions is maintained to be high.

Fig. 4: Y2H Protocol Visual. The image above shows the basic schematic of the two-hybrid system. The graph demonstrates the prey and the bait proteins in relationship to the activation domain (shown as Act) [6 Van Criekinge W, Beyaert R. Yeast Two-Hybrid: State of the Art." Biol Proced Online. 1999;2(1-38) .].