Yeast hybrid system

The three hybrid system of yeast has been described in this ppt. Yeast one Hybrid system, yeast two hybrid system and yeast 3 hybrid system is explained. This explain about the DNA-protein interaction and Protein-DNA-Protein interaction.

1. Yeast Hybrid Systems Dr.N.C.J.Packia Leskhmi Allied Health Sciences NICHE

2. In Y2H experiments, the close physical interaction between two proteins (the bait and the prey) brings together the DNA-binding and activation domains of a transcription factor. This forms a functional transcriptional unit that turns on expression of a reporter gene. In a Y1H system, detection is based on the interaction of a single protein (the prey) with a bait DNA sequence positioned upstream of a reporter gene. Importantly, the prey protein is fused to a transcriptional activation domain. Positive protein-DNA interactions bring the fusion activation domain into close proximity with the promoter element, thus activating downstream transcription of the reporter gene

3. Y1H assay has some clear advantages: Unlike luciferase reporter assays, Y1H is able to detect protein-DNA interactions that are not actually activating transcription. Fusing your protein(s) of interest to a strong activation domain allows Y1H to detect a variety of DNA-binding proteins, including those that do not directly function in transcription, e.g. replication proteins, DNA repair proteins, and repressor proteins. Y1H is compatible with many existing libraries. Most Y1H experiments can use hybrid prey libraries that have been constructed for Y2H applications, e.g. Gal4p- or LexA -based protein libraries can also be used for screening against various DNA baits in Y1H. Y1H can detect isoform-specific interactions. You can design your prey plasmids to encode specific protein isoforms, allowing detection of highly specific interactions. In contrast, ChIP -based approaches rely on sensitive and specific antibody binding to your protein of interest, and these are rarely able to be achieved on an isoform-specific level.

4. Y1H has some disadvantages too: Y1H doesn’t provide any information about the functional consequences of a DNA-protein interaction. The rates of false-positive results are high, as either endogenous yeast transcription factors or prey proteins that can bind Gal4 promoters can activate transcription of the reporter gene without interacting with the bait DNA sequence. Expression of your prey protein fused to an activation domain can result in improper protein folding, resulting in steric hindrance and loss of interactions, and/or failure to correctly localise to the nucleus.

5. Yeast 2 Hybrid System

6. The yeast-2-hybrid system is a simple scientific technique used to screen a library of proteins for potential interactions Firstly, a transcription factor is broken into two parts – a DNA-binding domain (BD) and a catalytic activation domain (AD) The DNA-binding domain is fused to a protein of interest called the bait (e.g. an enzyme) The activation domain is fused to a number of potential binding partners – called the prey (e.g. different ligands) If the bait and prey interact, the two parts of the transcription factor are reconstituted and activate transcription of a gene If the bait and prey do not interact, the two parts of the transcription factor remain separate and transcription doesn’t occur

7. The yeast-2-hybrid system detects protein-protein interactions according to the activation of a reporter gene The reporter gene may encode for the production of a protein that causes a visible colour change (e.g. ß-galactosidase) Alternatively, the reporter gene may encode for the production of an essential amino acid that is required for the yeast to grow on a deficient media (hence yeast growth would indicate successful interaction between bait and prey) Yeast-2-hybrid screens are a simple technique and hence have a relatively high rate of false positives (partial interactions) Consequently, the yeast-2-hybrid system is typically only used as an initial test to identify possible protein interactions

8. Yeast 3 hybrid assay

9. The Functions of Y3H It is well-known that Y3H methods can be applied in a variety of research fields: a. Finding protein partners of a known RNA sequence from cDNA libraries b. Searching a natural RNA partner or ligand for a known RNA binding protein via RNA libraries c. Testing suspected RNA-protein interactors d. Mutational analysis of interacting RNA and protein e. Discovery of multiprotein-RNA complexes In addition, Y3H is still a sensitive method to screen for the interactions between small molecule drugs and their protein targets.

10. The Principle of Y3H Yeast three-hybrid system is a derivative of yeast two-hybrid (Y2H). It’s a kind of powerful tool to dissert RNA-protein interactions of interest and that typically consists of three chimeric components. The first hybrid protein is made up of an RNA binding protein (RBD) fused to a DNA binding domain (DBD). The second fusion protein molecule contains a second RNA binding protein fused to the transcriptional activation domain (AD). The third hybrid part is an RNA molecule which bridges above two fusion proteins by providing two specific RNA targets for the RNA binding proteins. When this tripartite constituent forms at a promoter, the reporter gene is turned on, even transiently. And the expressed reporter products can be recognized by simple biochemical or phenotypic assays.

11. Key Benefits of Y3H In an RNA Y3H method, there are lots of great advantages presented on Creative Biolabs’ platforms: Fast and sensitive approach to identify and characterize RNA-protein interaction Applicable to a wide range of RNA-protein interactions Detection of both very strong and very weak interactions of RNA and proteins Assay results monitored easily by cell growth, colony color, or the levels of a specific enzyme The species of yeast is an ideal model organism in eukaryotic genetics Technique improved to reduce the selection of false-positive clones

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