FIGURE 26.19 Two-Hybrid Analysis: Mass Screening by Mating
To identify all possible protein interactions using the two-hybrid system, haploid a yeast are transformed with the Bait library, and haploid a yeast are transformed with the Prey library. When the two yeast types are mated with each other, the diploid cells will each contain a single bait fusion protein and a single prey fusion protein. If the two proteins interact, they activate the reporter gene, which allows the yeast to grow on media lacking histidine (yeast His3 gene) or turns the yeast cells blue when grown on X-gal media (lacZ gene from E. coli). This process can be done for all 6,000 yeast proteins using automated techniques.
The RNA three-hybrid system identifies proteins that interact through an intermediary RNA molecule. Two fusion proteins are used, one (yellow/purple) includes the DBD and the other (green/red) includes the AD of the transcription factor. When these two fusion proteins interact via an RNA molecule, they activate the reporter gene.
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