Question

what is Yeast 2 Hybrid is and what is it used for? What are the bait and prey and the concept behind how the Y2H experiment works? What are the advantages/disadvantages to it?

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Yeast 2 Hybrid

Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

Yeast 2 Hybrid used

Split-ubiquitin yeast two-hybrid. One limitation of classic yeast two-hybrid screens is that they are limited to soluble proteins. It is therefore impossible to use them to study the protein–protein interactions between insoluble integral membrane proteins.

1. Determination of sequences crucial for interaction-By changing specific amino acids by mutating the corresponding DNA base-pairs in the plasmids used, the importance of those amino acid residues in maintaining the interaction can be determined. After using bacterial cell-based method to select DNA-binding proteins, it is necessary to check the specificity of these domains as there is a limit to the extent to which the bacterial cell genome can act as a sink for domains with an affinity for other sequences (or indeed, a general affinity for DNA).
2. Drug and poison discovery-Protein–protein signalling interactions pose suitable therapeutic targets due to their specificity and pervasiveness. The random drug discovery approach uses compound banks that comprise random chemical structures, and requires a high-throughput method to test these structures in their intended target. The cell chosen for the investigation can be specifically engineered to mirror the molecular aspect that the investigator intends to study and then used to identify new human or animal therapeutics or anti-pest agents.
3. Determination of protein function-By determination of the interaction partners of unknown proteins, the possible functions of these new proteins may be inferred. This can be done using a single known protein against a library of unknown proteins or conversely, by selecting from a library of known proteins using a single protein of unknown function.

What are the bait and prey and the concept behind how the Y2H experiment works

The yeast two-hybrid system originally created by Fields and Song is a genetic system wherein the interaction between two proteins of interest is detected via the reconstitution of a transcription factor and the subsequent activation of reporter genes under the control of this transcription factor .

1. A protein X is expressed as a fusion to a DNA binding domain (DBD). The DBD–X fusion is commonly termed the “bait.” Because of the affinity of the DBD for its operator sequences the bait is bound to a promoter element upstream of a reporter gene but does not activate it because it lacks an activation domain.
2. A second protein Y is expressed as a fusion to an activation domain (AD) and is commonly termed the “prey.” They prey is capable of activating transcription but usually does not do so because it has no affinity for the promoter elements upstream of the reporter gene. If bait and prey are co expressed and the two proteins X and Y interact, then a functional transcription factor is reconstituted at the promoter site upstream of the reporter gene.
3. Consequently, transcription of the reporter gene is activated. Thus, in a yeast two-hybrid assay a protein–protein interaction is measured through the activation of one or several reporter genes in response to the assembly of a transcription factor by the said protein–protein interaction

Advantages and Disadvantages

Advantage

Ø The identity of the arrayed proteins is known, such that it is not necessary to isolate and sequence the library insert.

Ø The absence of fusions that are in the wrong reading frame or correspond to non-coding DNA avoids interaction signals from non-natural peptides.

Ø The library is normalised with respect to the representation of each protein. This is in stark contrast to classical cDNA libraries, in which cDNAs from highly expressed mRNAs are overrepresented and cDNAs from lowly expressed mRNAs are rare, which results in difficulties to find interactions with lowly expressed mRNAs.

Disadvantage

Created by the cloning of pools of DNA fragments is the uncontrolled fashion in which the coding sequences of the inserts are attached to the coding sequence of the split transcription factor.