The yeast two-hybrid screening system is a technique used for identifying molecular interactions and characterizing interaction pairs.
Credit: Tobias Arhelger/Shutterstock.com
The two-hybrid screening method was originally based on the DNA binding domain of the transcription factor GAL4 from the yeast Saccharomyces cerevisiae , which is incapable of activating transcription unless it is associated with an activating domain.
The two domains must pair to begin DNA transcription. Two-hybrid screening may be used to study protein-protein interactions, DNA-DNA interactions, or protein-DNA interactions.
How it works
The method was invented by Stanley Fields and Ok-Kyu Song in 1989. They designed an assay to detect protein-protein interactions using GAL4 to activate transcription of enzymes related to galactose utilization.
Two-hybrid screening can be carried out as a benchtop assay, a high-throughput screen, or a library screen.
The two-hybrid system has the advantage that it is an in vivo technique, meaning it is carried out in a live yeast cell. This offers more faithful representation of eukaryotic cellular biology. Another advantage is that it can track weak or transient interactions, because the reporter gene strategy results in an amplification. On the other hand, this causes a higher number of false positives.
Applications
The two-hybrid system has been used to study molecular interactions in all parts of the cell, including the membrane, mitochondria, cytoplasm, and nucleus. It also has applications in many different species of plants, animals, and microorganisms.
Interactome mapping :
Two-hybrid assays have been used not only to study individual molecular interactions, but to reveal larger scale behaviors and properties of cells. For example, in one recent study, two-hybrid technology was used to study cell polarity. Cell polarity is the asymmetric distribution of components and functions in a cell.
Polarity is critical in the development of different cell types through asymmetric cell divisions. It is established through a network of protein interactions that is not yet well understood.
A map of all interactions in a cell or organism is known as an interactome. Scientists mapped the polarity interactome using the two-hybrid system to generate proteome-wide interactome maps that significantly advanced understanding of cellular polarity.
Host-parasite interactions :
Another novel application makes use of the two-hybrid system to screen and identify host proteins interacting with Toxoplasma gondii Rhoptry Protein ROP16.
Toxoplasma gondii is a parasite that is able to manipulate its host for survival. It secretes the ROP16 protein into the host cell, which then activates host STAT signaling pathway.The study sought to characterize other host protein interactions with ROP16, and successfully identified two previously unknown host protein interactions in mice.
Post translational modification in Arabidopsis :
Small ubiquitin-like modifier (SUMO) proteins carry out post-translational modifications of proteins involved in numerous cellular processes.
In a study involving Arabidopsis – a plant related to cabbage and mustard – researchers screened a yeast two-hybrid library of transcription factors that interact with the cell’s SUMO machinery. They identified 76 interactors from various transcription factor families.
Two-hybrid screening is a widely used and powerful tool for identifying and characterizing molecular interactions. It can be used at a small scale for studies of individual proteins, or on a larger scale for mapping hundreds of interactions or even an entire interactome. Its applications are diverse among different branches of life science and biomedical fields.
Y2H Screening Play
Credit: iGEM 2014 Team Goettingen: On the road to Boston/Youtube.com
Further Reading