Question

You are a researcher who has just discovered a new protein in a fungus. Design a yeast twohybrid experiment to identify the other proteins in the fungal cell with which your protein interacts, and explain how this could help you determine the function of your protein.

Short answer

Design a yeast two-hybrid experiment by preparing a bait construct with your protein, a prey cDNA library, and co-transforming yeast cells to identify protein interactions. Use these interactions to infer your protein's function.

Step-by-step solution

Understand the Yeast Two-Hybrid System

The yeast two-hybrid system is a molecular biology technique used to discover protein-protein interactions by testing for physical interactions between two proteins. This method involves using a transcription factor that is split into two separate proteins: one is fused to a "bait" protein and the other to a "prey" protein. If the bait and prey proteins interact, the transcription factor is reconstituted and can activate transcription of a reporter gene, indicating an interaction.

Prepare the Bait Construct

First, clone the gene encoding your newly discovered protein into a yeast two-hybrid vector, such that it is fused to the DNA binding domain of the transcription factor. This creates the 'bait' protein. Ensure that the cloning is done in a way that preserves the native conformation and functionality of your protein.

Prepare the Prey Library

Create a cDNA library from the fungal cell, wherein each cDNA is fused to the activation domain of the transcription factor. This library represents all potential interacting proteins ('prey') and will be screened for interaction with the bait protein.

Transform Yeast Cells

Co-transform the yeast cells with both the bait construct and the prey library. In each yeast cell, a different prey construct will be present alongside the bait construct, allowing for comprehensive screening of potential interactions.

Screen for Interactions

Culture the transformed yeast cells and screen for those that express the reporter gene. This expression indicates a successful protein-protein interaction between the bait and one of the prey proteins. Use selectable markers or reporter assays to identify yeast cells with these interactions.

Identify and Analyze Interacting Proteins

Recover the prey plasmids from yeast cells that showed reporter gene expression and sequence the cDNA inserts. This will identify the proteins that interact with the newly discovered bait protein. Further analysis, such as sequence or functional domain comparison, can help hypothesize the function of your protein based on its interacting partners.

Key concepts

Protein-Protein Interaction

Protein-protein interactions (PPIs) are essential for many biological processes. They are how proteins communicate and perform functions in living organisms. By interacting, proteins can form complex structures, regulate cellular functions, and carry out biological processes.
These interactions can be either stable or transient, based on the proteins' roles and the cellular context. Understanding which proteins interact with each other helps researchers infer the functions and pathways they are involved in.
With the yeast two-hybrid (Y2H) system, scientists can identify PPIs by linking protein interactions to an easily measurable reporter gene expression, making it a powerful tool in molecular biology.

Molecular Biology Technique

Molecular biology techniques are methods used to study and manipulate nucleic acids and proteins in cells. These techniques include cloning, sequencing, and expression analysis, which are foundational for research and application in biotechnology and medicine.
The yeast two-hybrid system is a classic example of a molecular biology technique used to study protein-protein interactions. It utilizes a simple eukaryotic organism - yeast - to express recombinant proteins and test their ability to interact.

• Uses a split transcription factor to detect interactions
• Involves cloning genes into vectors for expression in yeast
• Allows for easy screening of numerous potential interactions through a library

Understanding and employing such techniques are crucial for uncovering the roles of proteins in significant biological processes.

Gene Cloning

Gene cloning is the process of making multiple copies of a particular gene. In the context of the yeast two-hybrid system, it involves inserting the gene of interest into a vector that allows it to be expressed in yeast cells.
This process ensures that the protein is produced in a controlled environment where interactions can be studied.
Cloning the protein as a "bait" involves:

• Isolating the gene from the organism
• Using restriction enzymes or PCR to prepare the gene for insertion
• Ligating the gene into a vector that can express it alongside a DNA-binding domain

By cloning the gene correctly, researchers can maintain the protein’s native conformation and study its interactions efficiently.

cDNA Library

A cDNA library is a collection of cloned DNA sequences that represent the expressed genes of an organism. These libraries are critical tools for researchers looking to study gene expression or identify protein interactions.
In the yeast two-hybrid system, a cDNA library from a fungus would contain sequences that potentially encode proteins which can interact with the bait protein.

• Represents all the mRNA transcripts in the cell
• Expressed as protein fusions to test for interactions
• Allows screening of large numbers of possible interacting partners

Creating a comprehensive cDNA library is essential for ensuring that potential interactions are not missed in the screening process.

Reporter Gene Assay

Reporter gene assays are used to measure the activity of a specific genetic pathway by linking the expression to a detectable reporter. In the yeast two-hybrid system, the reconstitution of a transcription factor through protein-protein interaction leads to the activation of a reporter gene.
Common reporter genes include \(eta\)-galactosidase or green fluorescent protein (GFP), which produce easily measurable signals like color change or fluorescence.

• Indicates successful protein-protein interactions
• Allows quantification of interaction strength
• Can be used with selectable markers for specific identification

Utilizing reporter gene assays provides a robust and straightforward way to evaluate the presence and characteristics of interactions in the yeast two-hybrid system.