Used to look for protein-protein interactions
We know that our target proteins bind to several things but we dont know what exactly.
1) Create library in modified yeast cells (insert reporter construct binding site for DNA binding domain; plasmid (expression vector) that produces our bait
2) Fuse DNA binding domain to our bait protein
3) GEt a bunch of candidate proteins that bind to bait and modify so they have an epitope tag with TAD
extra: When this construct is introduced into yeast, the cells produce the target protein attached to this DNA-binding domain (Figure 8-51). This protein binds to the regulatory region of a reporter gene, where it serves as “bait” to fish for proteins that interact with the target protein inside a yeast cell. To prepare a set of potential binding partners, DNA encoding the activation domain of a gene activator protein is ligated to a large mixture of DNA fragments from a cDNA library. Members of this collection of genes—the “prey”—are introduced individually into yeast cells containing the bait. If the yeast cell has received a DNA clone that expresses a prey partner for the bait protein, the two halves of a transcriptional activator are united, switching on the reporter gene
- If bait and candidate bind, TAD brought to transcription point, where transcription is allowed tobegin
- If reporter gene is on, prey is the binding partner for bait