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Protein-Protein Interaction Screens

Protein-Protein Interaction Screens. target. Bacterial Two-Hybrid System. bait. DNA binding protein. RNA polymerase. selectable marker. target sequence. Yeast Two-Hybrid System. activation domain. transcription factor. DNA binding protein. target. Yeast Two-Hybrid System.

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Protein-Protein Interaction Screens

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  1. Protein-Protein Interaction Screens

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

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

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

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

  6. His3 as Selectable Marker and 3-AT

  7. 3-amino triazole (3AT) NH3 N N NH

  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 1 gene 2 gene 3 gene 4 gene 5 gene 6 Libraries for 2-Hybrid Screens X X X X X

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

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

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

  14. activation domain/polymerase subunit activation domain/polymerase subunit activation domain/polymerase subunit Library Construction

  15. Library Construction activation domain selectable marker target fragment

  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)

  18. 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?

  19. 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)

  20. 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

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

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