PROTEIN INTERACTIONS: THE YEAST TWO-HYBRID SYSTEM
In addition to protein function and expression, proteomics attempts to find relevant protein interactions. For those who like “-omics” terminology, the total of all protein-protein interactions is called the protein interactome. For example, hormones usually bind to receptors that pass the signal on. Often this involves a protein relay where one protein activates another, which in turn activates yet another. To understand hormone function, researchers must identify all the proteins in the signal cascade. Phage display is one way to identify interactions, but the displayed proteins may not fold correctly or specific cofactors may be missing when mammalian proteins are expressed in bacteria.
An approach to overcoming these difficulties is the yeast two-hybrid system, where the binding of two proteins activates a reporter gene. The binding of a transcriptional activator protein, GAL4, to the promoter region of the reporter gene activates transcription and translation. GAL4 contains two domains needed to turn on the reporter gene. The DNA binding domain (DBD) recognizes the promoter element and positions the second domain, the activation domain (AD) next to RNA polymerase, where it activates transcription. These two domains can be expressed as separate proteins, but cannot activate the reporter gene unless they are brought together (Fig. 9.21).
In the two-hybrid system, the two domains are each fused to different proteins by creating hybrid genes. The bait is the DBD genetically fused to the protein of interest, and the prey is the AD fused to proteins that are being screened for interaction with the bait. When the bait and prey bind, the DBD and AD activate transcription of the reporter gene.
Two vectors are needed to perform two-hybrid analysis (Fig. 9.22). The first vector has a multiple cloning site for the bait protein 3′ of the GAL4-DBD; therefore, the fusion protein has the Bait protein as its C-terminal domain. The second vector has a multiple cloning site for the Prey protein 5′ of GAL4-AD and the fusion protein has the Prey as its N-terminal domain. Both plasmids must be expressed in the same yeast cell. If the bait and prey proteins interact, the reporter gene is turned on.
The reporter genes must be engineered to be under control of the GAL4 recognition sequence. Common reporter genes include HIS3, which encodes an enzyme in the histidine pathway and whose expression allows yeast cells to grow on media lacking histidine, or URA3, which allows growth without uracil. These reporter systems require yeast host cells that are defective in the corresponding genes. However, they do allow direct selection of positive isolates.
Another reporter used is lacZ from E. coli, which encodes β-galactosidase. Both bacteria and yeast that express lacZ turn blue when grown with X-Gal. β-galactosidase cleaves X-Gal, releasing a blue product. The reporter genes are usually integrated into the yeast genome, rather than being carried on a separate vector.
The yeast two-hybrid system has been used to identify all the protein interactions in the yeast proteome by mass screening with mating (Fig. 9.23). Yeast has about 6000 different proteins, and each of these has been cloned into both vectors via PCR. This way, each protein can be used as both bait and prey. All the bait vectors were transformed into haploid yeast of one
mating type, and the prey vectors into the other mating type. Haploid cells carrying bait are fused to haploid cells with prey and the resulting diploid cells are screened for reporter gene activity. This analysis thus examined 6000 × 6000 combinations for protein interaction.
The yeast two-hybrid system has some limitations. Because transcription factors must be in the nucleus to work, the target proteins must also function in the nucleus. For some proteins, entering the nucleus may cause the protein to misfold. For other proteins, the nucleus does not have the proper cofactors and the protein may be unstable. Large proteins may not be expressed well, or may be toxic to the yeast, leading to false negative results.
Copyright © 2018-2020 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.