yeast-two-hybrid and related...

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