Yeast Two-Hybrid

Uses fusion proteins and a modular transcription factor (where 2+ parts are required to have a working transcription factor). Used to discover protein-protein and protein-DNA interactions.
  1. DNA binding domain is fused to ‘bait’
    The bait plasmid codes for the DNA binding domain of the transcription factor in the protein of interest
    It also codes for a selection marker (eg – for antibiotic resistance, or for biosynthesis of a specific amino acid that isn’t present in the growth medium)
  2. Transcription activation domain is fused to ‘prey’
    The prey plasmid codes for the activation domain of the transcription factor, and is fused to the protein of interest
    It also codes for a selection marker
  3. If the bait and the prey are intact, the reporter gene is transcribed
This process is used in screening programmes