yeast-two-hybrid and related...
TRANSCRIPT
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Yeast-two-hybrid and Related Screens
Advanced Genetics 2/23/15
Zuzana Kocsisova
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Proteins interact with other proteins
• Often act in complexes
RISC
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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
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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
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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
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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
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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.
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Y2H: using the split Gal4-UAS system to go “fishing” for protein-protein interactions Test many different “prey” e.g. cDNA library
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Creating a cDNA library
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Reporters
• Auxotroph -> prototroph selection – Amino acid biosynthesis (His, Leu, Ade, Ura) – Nucleic acid biosynthesis
• Color detection screen – LacZ: blue/white screen – GFP
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Yeast two-hybrid assay of different LexA-fusion proteins (baits) with various PDZ
domains (preys)
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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
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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
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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
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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
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Reverse Y2H
Something disrupts the interaction:
1
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Replica Plating
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Protein-RNA interactions: Yeast three-hybrid
Gal4BD-Protein1-RNA-Protein2-Gal4AD
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Protein-DNA interactions: Yeast-one-hybrid
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Questions?
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Separation-of-function edgetic alleles
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Modified Reverse Y2H
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Library construction for R-Y2H
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Illustration of ced-9 edgetic alleles
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Experimental Workflow
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Consequences of network perturbations
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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
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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
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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