yeast-two-hybrid and related...
TRANSCRIPT
Yeast-two-hybrid and Related Screens
Advanced Genetics 2/23/15
Zuzana Kocsisova
Proteins interact with other proteins
• Often act in complexes
RISC
How to identify protein-protein interactions?
• Affinity purification – mass spectrometry • Co-immunoprecipitation • Protein microarrays
• Yeast-two hybrid system • Split-ubiquitin system • Bacterial-two-hybrid system
Y2H: using the split Gal4-UAS system to go “fishing” for protein-protein interactions This is how Gal4 UAS normally works: UAS = Upstream activation sequence Gal4 = a transcription factor made up of a DNA binding and an activation domain
The DNA binding domain (BD) alone cannot activate transcription The “bait” is fused to the BD
The DNA binding domain and activation domains of GAL-4 can be separated
The activation domain (AD) alone cannot bind the UAS The “prey” is fused to the AD
The DNA binding domain and activation domains of GAL-4 can be separated
Y2H: using the split Gal4-UAS system to go “fishing” for protein-protein interactions If the “prey” binds to the “bait” the AD and BD are brought together and activate transcription of the reporter and an interaction is discovered.
Y2H: using the split Gal4-UAS system to go “fishing” for protein-protein interactions Test many different “prey” e.g. cDNA library
Creating a cDNA library
Reporters
• Auxotroph -> prototroph selection – Amino acid biosynthesis (His, Leu, Ade, Ura) – Nucleic acid biosynthesis
• Color detection screen – LacZ: blue/white screen – GFP
Yeast two-hybrid assay of different LexA-fusion proteins (baits) with various PDZ
domains (preys)
Host Organism • Yeast
– Eukaryotic – Hospitable internal environment – Genome sequenced, techniques to manipulate – Only able to detect interactions in the nucleus – Some proteins are toxic to yeast
• E. coli – Higher transformation efficiency – Do not need a nuclear localization signal – Can use strains without interfering methyltransferase
activity • Mammalian Cells
Benefits of Y2H
• Can detect transient interactions not found in co-IP – Semi-quantitative
• Can be used to study known interactions – Modify specific residues, observe whether
interaction is maintained
• Immediate identification of the gene that encodes the product
Weaknesses of Y2H
• False positives – Proteins may not be expressed together in reality – Unnatural concentrations – Non-specific interactions
• False negatives – Interactions in the nucleus – Fusion to AD or BD may block – Yeast may lack chaperones
Related Screens
• Reverse yeast-two hybrid – Detect when an interaction is disrupted
• Yeast three-hybrid – Detect Protein-RNA interactions
• Yeast one-hybrid – Detect Protein-DNA interactions
Reverse Y2H
Something disrupts the interaction:
1
Replica Plating
Protein-RNA interactions: Yeast three-hybrid
Gal4BD-Protein1-RNA-Protein2-Gal4AD
Protein-DNA interactions: Yeast-one-hybrid
Questions?
Separation-of-function edgetic alleles
Modified Reverse Y2H
Library construction for R-Y2H
Illustration of ced-9 edgetic alleles
Experimental Workflow
Consequences of network perturbations
References • Fields, S. and O.-k. Song (1989). "A novel genetic system to detect protein–
protein interactions." Nature 340(6230): 245-246. • Licitra, E. J. and J. O. Liu (1996). "A three-hybrid system for detecting small
ligand–protein receptor interactions." Proceedings of the National Academy of Sciences 93(23): 12817-12821.
• Vidal, M., R. K. Brachmann, et al. (1996). "Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions." Proceedings of the National Academy of Sciences 93(19): 10315-10320.
• Chong, J. A. and G. Mandel (1997). "Isolation of DNA-binding proteins using one-hybrid genetic screens." The Yeast Two-Hybrid System: 289-297.
• Gubler, U., & Hoffman, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene, 25(2), 263-269.
• The yeast two-hybrid system edited by Paul L. Bartel and Stanley Fields. Published by Oxford University Press, 198 Madison Avenue, New York, New York 10016, USA; 1997. http://www.nature.com/nsmb/journal/v5/n7/full/nsb0798_535.html
Sources
• http://www.slideshare.net/25071987/yeast-two-hybrid-9235978
• http://www.sumanasinc.com/webcontent/animations/content/yeasttwohybrid.html
• http://en.wikipedia.org/wiki/Two-hybrid_screening
• http://en.wikipedia.org/wiki/CDNA_library • http://en.wikipedia.org/wiki/Muller%27s_mor
phs
Image Sources
• http://www.nature.com/nsmb/journal/v16/n11/fig_tab/nsmb.1673_F4.html
• http://openi.nlm.nih.gov/imgs/512/109/2570519/2570519_6604636f1.png
• http://www.cell.com/cms/attachment/2002982446/2011316676/gr2.jpg
• http://upload.wikimedia.org/wikipedia/commons/6/61/Two_hybrid_assay.svg
• http://www.nature.com/onc/journal/v22/n18/fig_tab/1206359f3.html