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Methods for a Yeast Three Hybrid System

Replication of chromosomes, messenger RNA processing, translation of proteins, and critical decisions in early stages of development depend on the interaction of DNA, RNA and proteins. These interactions are the basic biological processes of gene expression and protein synthesis. Scientists often use Saccharomyces cerevisiae, the same species of yeast used in baking and brewing, to study the genes that code for specific proteins and analyze the interactions between proteins and other molecules. The yeast three-hybrid system is one of three variants of the yeast two-hybrid system used to study these interactions.

The Two-Hybrid System
- The two-hybrid method is used to detect protein-protein interactions by looking for the expression of a reporter gene. Many proteins have at least two domains, or discrete structural and functional units. GAL4, a protein found in Saccharomyces cerevisiae, has a DNA binding domain (DBD) and an activation domain (AD). In this method, one hybrid protein is fused to the DBD and a second to the AD. No reporter gene is expressed by the fusion of only one of the hybrids. Since the GAL4 reporter gene is only expressed when both hybrids are present, protein-protein interaction is indicated.
The Three-Hybrid System
- The three-hybrid method identifies a reporter gene to detect the interaction of protein with RNA. The first hybrid protein consists of a DBD fused directly to an RNA binding domain. The second consists of the AD of the GAL4 transcription factor linked to an RNA binding protein. The RNA binding domains interact with a hybrid molecule of RNA that binds the two hybrid proteins to form a complex. Formation of the complex triggers the expression of the GAL4 reporter gene. Since the reporter gene is only turned on by the formation of the three-hybrid complex, its presence indicates the interaction of RNA and protein.
The One-Hybrid System
- The one-hybrid method is used to detect the interaction of protein with DNA. A one-hybrid system fuses a single protein, which links the AD directly to the DBD. Instead of identifying a reporter gene, this method looks for a specific protein that must recognize a target sequence of DNA. The presence of the specific protein indicates the interaction of protein with DNA.
The One-Two Hybrid System
- The one-two hybrid method is used to study protein-protein and protein-DNA interactions simultaneously. Using newly developed yeast strains, this method employs the GAL4 reporter gene to identify protein-protein interactions and a p53 consensus to identify protein-DNA interaction. The method is expected to significantly increase the precision of experimental results by reducing the occurrence of false positives obtained in other hybrid systems.

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