1. Protein-Protein Interaction Screens

2. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

3. Yeast Two-Hybrid System DNA binding protein activation domain transcription factor

4. Yeast Two-Hybrid System transcription factor activation domain DNA binding protein bait target

5. Yeast Two-Hybrid System selectable marker RNA polymerase transcription factor binding site DNA binding protein bait activation domain target

6. His3 as Selectable Marker and 3-AT

7. 3-amino triazole (3AT) NH N N NH 3

8. Libraries for 2-Hybrid Screens The goal of a two-hybrid experiment is to screen a “library” for proteins that interact with a bait protein of interest The library is a collection of plasmids that each contain a DIFFERENT TARGET PROTEIN fused to an activation domain or polymerase subunit

9. Libraries for 2-Hybrid Screens

10. gene 1gene 2gene 3gene 4gene 5gene 6 XXXXX

11. Libraries for 2-Hybrid Screens activation domain/polymerase subunittarget protein Target protein must be in same reading-frame as activation domain and not contain non-coding sequence (5’ UTR, introns)

12. Library Construction target gene genome PCR primers activation domain/polymerase subunit target gene

13. cDNA Library Construction target gene (coding region) mRNA primer cDNA fragment

14. Library Construction activation domain/polymerase subunit

15. Library Construction target fragment selectable marker activation domain

16. Whole-Genome Two-Hybrid Screens Construction of Bait and Target Libraries Covering Entire Proteome Automated Screening for Positives

17. Yeast Two-Hybrid Screen (Uetz et al., Nature 2000)

19. Problems with Two-Hybrid Screens While two-hybrid screens can be very useful, they suffer from fairly high false- negative and false-positive rates What are some potential sources of false- positive (proteins that appear to interact in the assay, but don’t in living cells) and false- negative (proteins that interact in living cells but not in the two-hybrid assay) results?

20. Two-Hybrid False Negatives Target protein not in library Proteins do not fold properly or interact in the conditions used in the screen (e.g. human proteins in yeast cells) Proteins only interact in the presence of other proteins Proteins interact in ways that do not permit activation domain to function (multimerization)

21. Two-Hybrid False Positives Non-specific –Bait proteins that activate without target –Target proteins that activate without bait –Target/Bait proteins that are “sticky” and interact with many things Specific –Interactions between proteins that are never expressed together in living cells –Interactions between proteins that are normally inhibited by the presence of other proteins/conditions

23. Other Methods to ID Protein-Protein Interactions Co-expression studies Structure prediction Purification of Complexes and Mass-Spec