Protein-protein interactions are closely associated with many biological processes, such as transcription, DNA modifications, and transcriptional regulations. Exploring the interactions between macromolecules contributes to revealing the molecular mechanisms of life processes. At Creative Biolabs, our experienced team of experts has built a reputation for providing cost-effective yeast three-hybrid (Y3H) services with fast turnaround times. Our comprehensive services range from initial experimental design to final analysis of results, helping you make breakthroughs in complex protein interactions.

Background of Y3H

Yeast hybrid systems are widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein-protein, protein-DNA, and protein-RNA interactions. For example, the yeast two-hybrid system (Y2H) plays a crucial role in studying protein-protein interactions. Y3H, an extension of the Y2H, is a powerful tool for studying more complex macromolecular interactions involving three components.

Principles of Y3H

Y2H system is a promising tool for detecting protein interactions. In addition to the case of two proteins interacting, it is also common for a third protein to stabilize the binding between the two partners. The Y3H system has gradually emerged as an attractive strategy to explore such complex protein complexes by introducing a new bait vector, pBridge, which allows to insertion of two exogenous proteins at the same time. In this system, a bait protein is expressed with a DNA binding domain (BD) and then the intermediate protein, and the prey protein is fused with the transcriptional activation domain (AD). The Pmet25 promoter in the pBridge vector only can be expressed in the medium lacking methionine. Thus, the interaction between bait protein and prey protein through the intermediate protein is identified in the medium lacking methionine after BD and AD vectors co-transformed into the yeast strain.

Fig.1 The yeast three-hybrid system vectors. (Sandrock, 2001)

Applications of Y3H

Similar to the Y2H system, the Y3H system can be applied in various research fields.

• Investigating more complex interactions between three proteins.
• Investigate the interaction between two proteins and a third protein by bringing the two hybrid proteins into proximity with each other by a third protein.
• The promoting or inhibiting effect of the third protein on the other two proteins was explored by the growth state of the colony.
• Analysis of polyprotein-RNA complexes.
• Screening for the interactions between small molecule drugs and protein interaction.

Features of Y3H

• The Y3H system is easy to operate.
• The third component is diverse and can be a protein, RNA, or small molecule.
• Applicable to characterize protein-protein and protein-RNA interaction.
• Effective detection of weak interactions between RNA/ protein and protein.

As a global company that places great emphasis on the fast delivery of solutions and cost-effective service, Creative Biolabs is committed to providing customized Y3H services for your specific project needs. Please contact us to tell us your needs, we will offer you flexible and innovative solutions and the highest quality Y3H services to help you solve the problems you encounter during your research.

Other optional protein-protein interaction (PPI) assay services:

• Two Hybrid Services
• Protein-Fragment Complementation Assay (PCA) Service
• Proximity-Dependent Biotin Identification (BioID) Service
• Protein-Protein Interaction Assay by X-Ray Crystallography Service
• Mammalian Protein-Protein Interaction Trap (MAPPIT) Service
• Magic™ Conversable Mammalian Library (CML) Platform for Biomolecular Interaction Discovery
• TurboID
• Y2H-Based Drug-Target Interaction Identification

Published Data

• Identification of Host Factors Binding to DENV and ZIKV Virus Subgenomic RNA by Y3H Screening

Fig. 2 Detecting RNA-binding proteins in a mating yeast three-hybrid ORF screen. (Sander Jansen, 2021)

In the process of infection, extracellular ribonuclease XRN1 can not completely degrade the RNA genomes of dengue virus (DENV) and Zika virus (ZIKV), which will lead to the formation of small subgenomic flavivirus RNA (sfRNA) in infected host cells. These non-coding RNAs are key virulence factors, which always seem to affect the molecular interaction with RNA-binding proteins (RBP) in the cellular process. Here, the researchers used the yeast three-hybrid (Y3H) system for correlation analysis. They improved the RNA-Y3H method to expand the proteome screening of RBP. Through the optimized Y3H system, they screened human proteins bound to DENV and ZIKV sfRNA and obtained a list of 69 presumptive sfRNA conjugates, including several previously reported conjugates and many novel RBP host factors.

FAQ

1. What is the Yeast Three-Hybrid (Y3H) System and How Does it Work?

   The yeast three-hybrid (Y3H) system is an adaptation of the yeast two-hybrid system that is designed to detect and analyze interactions between two proteins and a third molecule, typically an RNA or a small molecule. In this system, one protein is fused to a DNA-binding domain (DBD) and the other to an activation domain (AD). The third component, such as RNA, is linked to a molecule that can bridge the DBD and AD fusions when bound by the two proteins. If all three components interact successfully, the reconstituted DBD and AD drive the transcription of a reporter gene, indicating a positive interaction.

2. What are the Advantages of Using the Y3H System in Research?

   The Y3H system is particularly useful for studying the interactions that involve a third component, such as RNA molecules or small biochemical molecules. This system allows researchers to:

   • Identify and characterize novel interactions involving small molecules or RNA that might not be detectable by other methods.
   • Explore the effects of small molecules on protein-protein interactions, which can provide insights into their mechanism of action and potential therapeutic applications.
   • Investigate post-translational modifications and their influence on the interactions between proteins and other molecules, enhancing our understanding of cellular regulation and signaling pathways.
3. How Can the Y3H System Be Applied in Drug Discovery and Development?
   • Target Validation: By confirming interactions between a potential drug target and other proteins in the presence of a candidate drug molecule, researchers can better understand the mechanism of action of the drug.
   • High-Throughput Screening: The Y3H system can be adapted for high-throughput screening to identify molecules that disrupt or enhance specific protein interactions, which is valuable for identifying potential therapeutics.
   • Pathway Analysis: By using the Y3H system, researchers can map interaction networks that involve small molecules, helping to elucidate pathways affected by drugs and identify potential off-target effects or synergistic targets.
4. How does the yeast three-hybrid system differ from the yeast two-hybrid system?

   The yeast three-hybrid (Y3H) system is an extension of the yeast two-hybrid (Y2H) system, which is traditionally used to study protein-protein interactions. The Y3H system incorporates a third component, typically an RNA molecule or a small chemical compound, allowing for the investigation of the interaction between two proteins in the presence of this third molecule. This additional component can be critical for interactions that are dependent on or regulated by small molecules or RNA, providing a more nuanced understanding of protein dynamics and function within the cell.

5. Can the yeast three-hybrid system be used to study interactions involving DNA or other biomolecules?

   Yes, the yeast three-hybrid system can be adapted to study a wide range of interactions involving various biomolecules, including DNA, peptides, and even certain post-translational modifications. By modifying the third component to include these different molecules, researchers can explore how these elements influence protein interactions. This flexibility makes the Y3H system a versatile tool for studying complex biochemical pathways and molecular interactions that are critical to cellular function and disease processes.

6. What types of reporter genes are commonly used in the Y3H system, and why are they important?

   Commonly used reporter genes include HIS3, lacZ, and ADE2.

   • HIS3 allows for growth on histidine-deficient media, providing a selection marker for positive interactions.
   • lacZ encodes β-galactosidase, which can be assayed colorimetrically to measure the strength of interactions.
   • ADE2 expression results in growth on media lacking adenine and can produce a color change in colonies depending on metabolic activity.
7. Can the yeast three-hybrid system be used in quantitative analysis of molecular interactions?

   By quantifying the expression level of the reporter gene, researchers can gauge the strength and stability of these interactions between proteins and the third component under different conditions. This quantitative approach can be enhanced by using fluorescent or luminescent reporters, which allow for real-time, dynamic measurement of interaction strengths. Such quantitative data are invaluable for understanding the kinetics and binding affinities involved in molecular interactions, aiding in detailed mechanistic studies and the design of therapeutic interventions.

8. What are the potential applications of the Y3H system in functional genomics and proteomics?
   • Interaction Mapping: It can be used to map interaction networks involving RNA molecules or small molecules, helping to elucidate the functional roles of these interactions in cellular processes.
   • Variant Analysis: By studying how genetic variants affect protein interactions in the presence of a third component, researchers can gain insights into disease mechanisms and identify potential therapeutic targets.
   • Protein Function Annotation: The Y3H system can help assign functions to uncharacterized proteins by identifying their interaction partners and the conditions under which these interactions occur, thereby filling in gaps in proteomic databases.

Resources

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