Introduction
Protein-DNA and protein-protein interactions are crucial in gene transcription, DNA replication, recombination, and repair. Transcription factors interact with promoters and enhancers to execute transcription and it is important to identify them to determine their DNA binding specificities and affinities (Sambrook et al.). Approximately 5-10% of all eukaryotic genes encode transcription factors (Reece-Hoyes et al. 2005; Kummerfeld and Teichmann 2006; Vaquerizas et al. 2009). Yeast two-hybrid (Y2H) systems / Yeast Two-Hybrid Assay can be used to study and characterize these protein-protein and protein-DNA interactions in living yeast cells. The most commonly used systems to study viral protein interactions are GAL4-based and LexA-based systems [3].
Key Components of yeast two-hybrid system
- Plasmid constructs containing DNA binding domain and activation domain of the transcription factor fused with desired proteins
- Auxotrophic yeast strain
- Regulated reporter constructs such as LEU2 or HIS3.
- The second reporter is the Lac Z gene.
Principle of the yeast two-hybrid system
The yeast two-hybrid system
A eukaryotic transcriptional factor has two domains namely, a DNA binding domain which is responsible for the specificity of the promoter sequence, and an activation domain which binds to the RNA polymerase and activates transcription.
These two domains are independent of each other i.e. a DNA binding domain can be bound to any heterologous activation domain and can still be functional in the sense that it can activate transcription of an appropriate corresponding promoter. Since the specificity is due to the DNA binding domain as it binds to the promoter sequence, DBD becomes the deciding factor of which gene would be transcribed.
The proteins whose interaction we want to study are expressed as DBD or AD fusion proteins (chimeric proteins). DBD-fusion protein is called bait and AD fusion protein is considered prey. When these fusion proteins interact, DBD and AD are brought together and can initiate the transcription of the gene construct containing the reporter gene.
The activation domain can be engineered according to the requirement which means that it can be optimized for making the interactions stronger or weaker.
Hence, this system can be used to screen the proteins (prey) that can possibly interact with the DBD-fusion protein (bait).
Commonly used is a GAL-4-based two-hybrid system in which genes for GAL 80 (GAL 4 binding protein) and wild-type GAL 4 have to be deleted because we want only chimeric proteins to be expressed. Alternative versions use the bacterial LexA DBD and VP16 AD (Golemis and Khazak 1997).
Pipeline for Y2H library screening
- A plasmid construct containing the DNA binding domain fused to the bait protein is transformed into an auxotrophic strain of yeast.
- Autoactivation of the DNA binding domain is checked and bait is verified.
- The activation domain-prey library is transformed into the bait strain.
- Auxotrophic yeast cells are plated on media lacking, say, histidine. Only yeast cells that are able to synthesize histidine viz transcription will be able to grow on the minimal media.
- Perform a beta-gal assay on the positives. This will be a reassurance.
- Determine prey identity by PCR/sequencing.
False positives
A major reason for a false positive result is the interaction of two proteins due to their forced presence in a system in an unnatural way whereas they may never encounter each other naturally. Fusion proteins usually show a high level of non-specific interactions. Bait protein may directly activate the reporter gene.
Reverse hybrid
As described earlier, the Yeast Two-Hybrid (Y2H) system is used to detect and characterize direct protein-protein interactions in living yeast cells. Building upon this classical approach, the Reverse Yeast Two-Hybrid system identifies regulatory proteins capable of modulating specific protein-protein interactions, thus providing new insights into the dynamic control of protein complexes. The reverse two-hybrid system is a powerful method to select mutations that disrupt and dissociate the interaction between two proteins and therefore to identify the residues involved in this interaction (Vincent et al., 2020) [4]
Comparative Analysis of Y2H and Reverse Y2H Systems
| Feature | Yeast Two-Hybrid (Y2H) | Reverse Yeast Two-Hybrid (RY2H) |
|---|---|---|
| Basic Principle | Interaction between bait and prey proteins activates reporter genes, enabling growth on selective media or producing color reaction (Fields & Song, 1989) | Facilitate identification of events that dissociate protein protein interactions. White, M. A. (1996) |
| Selection Strategy | Positive selection: Growth occurs when proteins interact | Negative selection: Growth occurs when interactions are disrupted |
| Primary Applications | • Identification of novel protein-protein interactions • Mapping protein interaction networks • Validation of predicted interactions • Library screening for binding partners (Brückner et al., 2009) | • Identification of interaction inhibitors • Mutation analysis affecting protein binding • Drug discovery for protein-protein interaction targets • Mapping interaction interfaces (Reverse & Young, 2002) |
| Reporter System | Typically uses auxotrophic markers (HIS3, LEU2) or colorimetric reporters (lacZ) for positive growth selection | Uses counter-selectable markers (URA3/5-FOA or CYH2) for negative growth selection (Boeke et al., 1987) |
| Screening Output | Identifies proteins that interact with bait protein | Identifies conditions or mutations that prevent protein interactions |
| Technical Advantages | • High sensitivity for weak interactions • Suitable for high-throughput screening • In vivo detection system • Cost-effective (Ferro & Trabalzini, 2013) | • Directly identifies interaction inhibitors • Useful for drug screening • Can identify interaction-specific mutations • Suitable for structure-function studies (Young et al., 1998) |
| Limitations | • False positives from spontaneous activation • Requires nuclear localization • May miss transient interactions • Not suitable for membrane proteins (Rajagopala et al., 2012) | • Higher technical complexity • Potential toxicity of counter-selectable markers • May require optimization of selection conditions • Limited to known interactions (Vincent et al., 2020) |
| Main Research Uses | • Interactome mapping • Protein function discovery • Validation of computational predictions • Disease-related protein studies (Yu et al., 2008) | • Drug development • Protein engineering • Structure-function analysis • Therapeutic target validation (Huang & Bader, 2009) |
| Validation Requirements | Secondary protein-protein interaction assays (co-IP, pull-down) | Biochemical confirmation of interaction disruption and specificity tests |
Key References:
- Fields, S., & Song, O. (1989). Nature, 340(6230), 245-246.
- Vidal, M., et al. (1996). Proceedings of the National Academy of Sciences, 93(19), 10315-10320.
- Boeke, J. D., et al. (1987). Methods in Enzymology, 154, 164-175.
- Young, K., et al. (1998). Molecular and Cellular Biology, 18(4), 1935-1945.
- Vincent, M., et al. (2020). Methods in Molecular Biology, 2196, 1-14.
References
- Reece-Hoyes, J. S., & Walhout, A. J. (2018). Gateway-compatible yeast one-hybrid and two-hybrid assays. Cold Spring Harbor Protocols, 2018(7), pdb-top094953.
- Kolanus, W. (1999). The two hybrid toolbox. Combinatorial Chemistry in Biology, 37-54.
- Guo, D., Rajamäki, M.-L., & Valkonen, J. (2008). Protein-Protein Interactions: The Yeast Two-Hybrid System. Methods in Molecular Biology™, 421–439. doi:10.1007/978-1-59745-102-4_29.
- White, M. A. (1996). The yeast two-hybrid system: forward and reverse. Proceedings of the National Academy of Sciences, 93(19), 10001–10003. https://doi.org/10.1073/pnas.93.19.10001/
- Vincent, O., Gutierrez-Nogués, A., Trejo-Herrero, A., & Navas, M. (2020). A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein–protein binding. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-77992-1
