Yeast two hybrid system

Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.

1. Experimental technique  Yeast two-hybrid system to identify Protein-Protein interactions

2. Protein-Protein interactions Protein–protein interactions are the physical contacts of high specificity, established between two or more protein molecules. Proteins control and mediate many of the biological activities of cells by these interactions. Information about PPIs improves our understanding of diseases and can provide the basis for new therapeutic approaches. Aberrant PPIs are the basis of multiple aggregation-related diseases, such as Creutzfeldt–Jakob, Alzheimer's disease, and may lead to cancer.

3. Examples of protein-protein interactions Signal Transduction: activity of cells is regulated through this way in form of extracellular signals Transport across cell membrane: a protein may be carrying another protein Cell metabolism: In many metabolic pathways different proteins interact to perform a specific function. Muscles contraction: myosin filaments act as molecular motors and by binding to actin enable filament sliding.

4. What is Yeast Two Hybrid system? Also known as Y2H or Two-hybrid screening molecular biology technique Two Hybrid system is for Isolating and Studying Protein-Protein Interactions. This system uses a reporter gene for detecting the interaction of pair of proteins in yeast cell nucleus. Y2H assay relies on the expression of a reporter gene (such as lacZ or GFP), which is activated by the binding of a particular transcription factor. 

5. Why Yeast Two Hybrid system The yeast two hybrid system has a clear advantage over classical biochemical or genetic methods It is an in vivo technique that uses the yeast cell as a living test-tube. It bears a greater resemblance to higher eukaryotic systems than a system based on a bacterial host. With regards to classical biochemical approaches, which can require high quantities of purified proteins or good quality anti- bodies, the two Hybrid system has minimal requirements to initiate screening, since only the cDNA of the gene of interest is needed.

6. How Yeast Two Hybrid system works? The DNA sequence that encodes target protein, is fused with the DNA that encodes the DNA binding domain of gene activator protein using recombinant DNA technology.  Interactions are identified by the transcription of reporter genes. Positives are selected using differential media.

7. The classical Yeast Two Hybrid system T he early yeast two-hybrid systems were based on the finding that many eukaryotic transcription factors have separable DNA-binding and transcription activation

8. The protein of interest, the “bait”, is fused to a DNA-binding domain. Proteins that bind to bait, the “fish” or “prey”, are fused to a transcription activation domain

9. Proteins that do not bind to the bait will not activate the transcription of the reporter gene Any protein that binds to the bait will activate the transcription of the reporter gene

10. The first step is to construct a bait plasmid and a library. Each type of plasmid contains a selectable marker such as an essential amino acid.

11. Advantages Immediate availability of the cloned gene of the interacting protein Only a single plasmid construction is required Interactions are detected in vivo Weak, transient interactions can be detected Can accumulate a weak signal over time Protein purification not necessary No antibodies requires

12. Disadvantages False positives are the largest problem with the yeast two-hybrid system. Can be caused by the ability of bait to induce transcription without interaction with the bait Possible incorrect protein folding in yeast gene encoding target protein must be available failed to detect some know interactions Elimination of False Positives Sequence Analysis Retransformation of both strain with bait plasmid and strain without bait plasmid Test for interaction with an unrelated protein as bait

13. Conclusion: The predictive power of the interactome model allows us to examine the organization and coordination of multiple complex cellular processes and determine how they are organized into pathways. The interactome model can be used to predict poorly characterized proteins into their functional context according to their interacting partners within a module. One-to-many relationship can be used for pathway discovery.