The Yeast Three-Hybrid (Y3H) system is an advanced variation of the Yeast Two-Hybrid (Y2H) assay designed to study interactions between two proteins mediated by a third molecule. It is commonly used to investigate protein-protein interactions that depend on small molecules, such as RNA, DNA, or small ligands. This system provides a way to study more complex molecular interactions and can give insights into regulatory mechanisms that require an intermediary molecule.

How the Yeast Three-Hybrid System Works:

1. Bait Protein:
The bait protein is fused to the DNA-binding domain (DBD) of a yeast transcription factor, just like in the traditional Y2H assay.

2. Prey Protein:
The prey protein is fused to the activation domain (AD) of the yeast transcription factor.

3. Mediator Molecule (Third Component):
The third molecule can be RNA, a small ligand, or another protein that mediates or stabilizes the interaction between the bait and prey proteins.
In RNA-mediated Y3H, for example, the RNA binds both the bait and prey proteins, bringing them into proximity to reconstitute the transcription factor.

4. Reconstitution of Transcription:
If the bait and prey proteins interact in the presence of the mediator molecule, the DBD and AD are brought together, activating the transcription of a reporter gene (like HIS3 or lacZ).
The reporter gene’s activation allows yeast cells to grow on selective media or exhibit a detectable signal, such as a color change.

Specific Applications of the Yeast Three-Hybrid System:

1. RNA-Protein Interactions:
One of the primary uses of Y3H is to study interactions between proteins and RNA molecules. For example, the bait protein may bind RNA, and the prey protein will interact with the RNA, revealing a protein-RNA-protein complex.
Y3H has been used to identify RNA sequences or motifs that mediate specific protein interactions.

2. Ligand-Dependent Protein Interactions:
The Y3H system can also study protein-protein interactions that depend on small ligands, such as hormones, metabolites, or drugs. The ligand acts as a bridge between the two proteins.
It helps in identifying proteins that interact only in the presence of a specific ligand, which is important for drug discovery and understanding signal transduction pathways.

3. Ternary Complex Formation:
The Y3H system allows researchers to study how two proteins interact only when a third component, such as a cofactor or another regulatory protein, is present.

Example of Yeast Three-Hybrid (RNA-Protein Interaction):

1. Bait Construct: A hybrid protein with a DBD fused to a protein that binds RNA (such as MS2 coat protein).
2. Prey Construct: A hybrid protein with an AD fused to the protein of interest that potentially interacts with RNA.
3. RNA Molecule: The RNA contains specific binding sites for both the bait and prey proteins. If the RNA mediates the interaction between the bait and prey proteins, the transcription factor is reconstituted, leading to reporter gene activation.

Advantages:
RNA-Protein Interaction Study: The Y3H system is a powerful tool for studying RNA-protein interactions, which are important in gene regulation, splicing, and RNA transport.
Ligand Dependency: It allows the exploration of protein interactions that occur only in the presence of specific small molecules or ligands.
Specificity: The presence of the third component (RNA or ligand) can increase the specificity of detected interactions, reducing false positives common in Y2H systems.

Limitations:
Complexity: The system is more complex than Y2H, requiring careful design of constructs and mediator molecules.
Mediator Requirement: Success depends on the proper folding and availability of the third component (e.g., RNA or ligand) in the yeast system.
False Negatives: The system may miss some interactions that do not rely on the mediator molecule or are transient in nature.

The Y3H system has been widely adopted in molecular biology to explore interactions beyond simple protein-protein pairs, especially for studying complex regulatory mechanisms involving RNAs or small molecules.
Reference source:
https://www.kmdbioscience.com/pages/yeast-three-hybrid-service.html