Two-hybrid screening

Two-hybrid screening is a molecular biology technique used to discover protein-protein interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule. It is a type of genetic screen. Two-hybrid screening originated from the fields of yeast genetics and molecular cloning, and has become a widely used method in molecular biology and biochemistry.

Overview

Two-hybrid screening involves the reconstitution of a functional transcription factor from two separate fragments, typically in a yeast cell. The technique is based on the principle that most transcription factors consist of two distinct domains: a DNA-binding domain (DBD) and an activation domain (AD). In the two-hybrid system, these domains are separated and individually fused to two proteins of interest. If the two proteins interact, the DBD and AD are brought into close proximity, reconstituting a functional transcription factor that can drive the expression of a reporter gene, indicating a positive interaction.

Components

• DNA-Binding Domain (DBD): This domain is responsible for anchoring the hybrid protein to a specific DNA sequence upstream of a reporter gene.
• Activation Domain (AD): This domain is necessary for activating transcription of the reporter gene.
• Reporter Gene: A gene whose product is easy to detect and measure, used to indicate the interaction between the two proteins of interest.
• Bait Protein: The protein of interest fused to the DBD. It "baits" the interaction.
• Prey Protein: The protein of interest fused to the AD. It is the "prey" in the interaction.

Applications

Two-hybrid screening is used in various applications, including:

• Identifying novel protein-protein interactions.
• Mapping interaction domains within proteins.
• Studying the effects of mutations on protein interactions.
• Screening libraries of proteins to identify interactors with a protein of interest.

Advantages and Limitations

Advantages:

• Can identify unknown protein-protein interactions.
• Allows for the study of protein interactions in a living cell context.

Limitations:

• False positives can occur, necessitating further validation of interactions.
• Not suitable for detecting interactions that require post-translational modifications or are transient in nature.
• Interactions are studied out of their natural context, which may affect their biological relevance.

Variants

Several variants of the two-hybrid system have been developed to overcome some of its limitations and to expand its utility:

• Reverse two-hybrid system: Used to screen for disruptors of protein-protein interactions.
• Three-hybrid system: Allows for the detection of interactions between two proteins and a third molecule, such as RNA or a small molecule.
• Membrane two-hybrid (MaTH): Adapted for identifying interactions between membrane proteins.

Conclusion

Two-hybrid screening is a powerful tool for exploring the interactome, the comprehensive map of protein-protein interactions within a cell. Despite its limitations, it has significantly contributed to our understanding of cellular processes and the molecular basis of diseases.