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Yeast Two-Hybrid
- 1.
- 2. Purpose: To determine directprotein-protein interactions
- 3. Background: Transcription Factors DNA-binding Domain Activation Domain Reporter Gene RNAPolymerase Transcription Factor
- 4.
- 5. Interaction of Bait andPrey Bait DNA-binding Domain Reporter Gene Prey Activation Domain RNA Polymerase
- 6.
- 7. Y2H Pros: Direct interactions Doesn’t require sophisticated equipment/machinery Can screen many proteins Can be automated Cons: Limited to soluble proteins Addressing transient nature of interactions False positives Protein fusion could inhibit some interactions Takes place in nucleus and proteins localized elsewhere may not show up in screen Some false positives may not be in the same cell at the same time All results should be confirmed by other techniques.
Editor's Notes
- #4 Transcription factors aid the cell in transcription of DNA. They bind specific DNA sequences upstream of genes and either repress or activate expression. Activating transcription factors consist of a DNA-binding domain which recognizes a specific sequence and an activating domain that recruits RNA polymerase to begin transcription.
- #5 The two transcription factor domains are functional when bound directly to one another and when bound indirectly. The yeast two-hybrid system requires the fusion of target proteins to each domain. Typically, a single known protein is used as bait and is bound to the DNA-binding domain. Oftentimes, a library of possible binding partners is fused to the activation domain and is used as prey. Construction of this library involves the ligation of cDNAs of interest to a plasmid. These plasmids and the bait plasmids each have selectable markers and are transformed into your cells and grown in selective media.
- #6 When these two fused proteins are expressed and able to bind with one another, the complex acts as the transcription factor and is able to recruit RNA polymerase to begin transcription of the reporter gene. If that interaction of bait and prey fails to occur, the reporter gene is not turned on.
- #7 Typically, the yeast strains used in Y2H systems are deficient in biosynthesis of certain nutrients—like histidine, for example. For this step, the transformed yeast cells are plated on selective media, perhaps histidine deficient media. Only those cells with direct protein-protein interaction of the bait and prey will have been able to grow because of the activation of His3, the reporter gene. Another reporter gene often used is lacZ. Those colonies showing the desired phenotype are subsequently PCRed and sequenced for the identity of the unknown protein.
- #8 (Wikipedia, Two-Hybrid Screening: Strengths and Weaknesses)