Yeast one-hybrid screening for DNA-protein interactions

Curr Protoc Mol Biol. 2001 Aug:Chapter 12:Unit 12.12. doi: 10.1002/0471142727.mb1212s55.

Abstract

One-hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with a specific regulatory DNA sequence of interest (the bait sequence). In this technique, the interaction between two proteins (bait and prey) is detected via in vivo reconstitution of a transcriptional activator that turns on expression of a reporter gene. Detection is based on the interaction of a transcription factor (prey) with a bait DNA sequence upstream of a reporter gene. To ensure that DNA binding results in reporter-gene activation, cDNA expression libraries are used to produce hybrids between the prey and a strong trans-activating domain. The advantage of cloning transcription factors or other DNA-binding proteins via one-hybrid screenings, compared to biochemical techniques, is that the procedure does not require specific optimization of in vitro conditions.

MeSH terms

  • DNA / metabolism*
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism*
  • Molecular Biology / methods*
  • Protein Binding
  • Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Two-Hybrid System Techniques*

Substances

  • DNA-Binding Proteins
  • Proteins
  • DNA