yeast two-hybrid system

What is the mechanism of yeast two-hybrid system?

-DNA binding domain fused to bait protein. -Bait then binds to UAS region -Activation domain fused to prey -binding the domain and fish to reporter gene

How do you make yeast two-hybrid system?

-Make a prey and bait plasmids with marker (amino acid) -Place both types of plasmids into yeast cells -Place yeast cell in amino acid deficient medium -place yeast cell in agar -Identify protein-protein interactions by sequencing plasmid DNA

What are the key players in yeast two-hybrid system?

-Upstream activating sequence -Reporter gene -DNA binding domain -Activating domain -Bait protein -Prey/fish protein -Transcription factor

What happens if the bait and prey proteins do interact?

The reporter gene will be transcribed into mRNA leading to a detectable phenotype

What is the goal of yeast two-hybrid system?

To discover protein-protein interactions

how is yeast two-hybrid system detected?

by the transcription of a reporter gene (growth of colony or color change)

How does yeast two-hybrid system discover protein-protein interactions?

through reconstitution of a transcription factor

What is the yeast two-hybrid system based on?

transcription initiation

What happens if the bait and prey proteins do not interact?

transcription will not occur