Yeast 2-Hybrid 2.26.18 Kayla Nygaard
Y2H - What is it? A method to screen for protein-protein interactions in yeast
Capitalizes on GAL4 system in yeast. GAL4 has 2 domains DNA-Binding Domain (DB) Transcriptional Activation Domain (AD) How does it work? https://www.sciencedirect.com/science/article/pii/b9780124201200000037
How does it work? These domains can be separated & fused to proteins of interest (X, Y) DB-X = bait AD-Y = prey https://www.sciencedirect.com/science/article/pii/b9780124201200000037
If X & Y interact, GAL4 domains will reunite and activate a reporter gene. How does it work? https://www.sciencedirect.com/science/article/pii/b9780124201200000037
Y2H: Possible Outcomes
Stop & Jot List 5 essential components in the Yeast 2-Hybrid assay. 30 seconds go!
Y2H Visual
Y2H: Reporters HIS3 Allows growth in the presence of HIS3 inhibitor 3AT LacZ turns colonies blue in the presences of X-gal
Caveats Learn as much as possible about X and Y Is the bait protein (X) known to activate transcription (or repress it?) Does bait or prey belong to a protein family? When & where are they expressed Will be necessary in construct cdna library How will you confirm interactions from screen? Co-IP of endogenous proteins
Y2H: Outcomes
CONSTRUCT DB FUSION PROTEIN How to Y2H: Step 1 Clone bait DNA fragment into plasmid pdbleu = GAL4 DNA-binding domain vector Via restriction enzyme digest + ligation May need to amplify bait DNA fragment with primers containing correct restriction sites Transform into competent E. coli Plate on kanamycin plates for selection Sequence at junction to confirm correct reading frame
CONSTRUCT AD FUSION PROTEIN How to Y2H: Step 2 Clone prey DNA fragments into plasmid ppc86 = GAL4 activation domain vector Same process as DB fusion Transform Plate on ampicillin plates Sequence **usually a library of cdna products
*TEST FOR SELF-ACTIVATION Measure HIS3 basal expression due to self-activation How to Y2H: Step 3 In both strains (pdbleu-x and ppc86-y) Increase 3AT in yeast media plates w/ histidine 3AT inhibits HIS3 in dose-dependent manner Titrate HIS3 activity until growth is inhibited Determine minimum effective dose Allows for weak protein-protein interactions to be detected For best results test with AD vector (minus prey)
Grow bacteria Harvest plasmid(s) How to Y2H: Step 4 Transform into yeast
How to Y2H: Step 5 Perform Screen
Replica Plating
Think About It Is there any way you could use a Y2H assay in your proposal? Why/why not?
Y2H: Options Traditional Assay http://mmg-233-2013-genetics-genomics.wikia.com/wiki/yeast_two-hybrid
Can detect transient interactions that might be missed in co-ip Semi-quantitative Can be used to study known interactions Modify residues, check if interaction is maintained Advantages Immediate identification of the gene that encodes the product By sequencing the plasmid from the colony of interest Can be scaled up easily or automated Low-tech no sophisticated equip. necessary Important first hint at interactions
Potential Weaknesses False positives Proteins may not be expressed together normally Unnatural concentrations Non-specific interactions False negatives Proteins may not localize to the nucleus Fusion to AD or BD may block Interaction may not occur in yeast May lack necessary chaperones so proteins unfolded Protein may miss necessary phosphorylation
Reverse Yeast 2-Hybrid Detect when an interaction is disrupted Yeast 3-hybrid Detect protein-rna interactions Y2H Adaptations Yeast 1-hybrid Detect protein-dna interactions Split-ubiquitin assay To detect membrane-bound protein interactions
Methods http://mmbr.asm.org/content/76/2/331/f5.large.jpg
Split-ubiquitin system to conquer membrane-bound proteins Other versions of Y2H CUB = c-terminal; NUB = n-terminal
Other versions of Y2H Incorporating co-factors:
Determining sequences crucial for interaction Applications Drug and Poison Discovery Determination of protein function Zinc finger protein selection
S. cerevisiae = original yeast used Candida albicans For C. albicans genes Other model systems E. coli = B2H Use larger libraries, lower false positives Mammalian two-hybrid = M2H Uses transfected cell lines Arabidopsis thaliana = P2H For plants! (protoplast two hybrid = P2H) Bombyx mori = I2H Insect two-hybrid
Will be posted later today. Read related papers if you haven t yet. HW Help with Dutcher HW today at 5pm in 6 th McKinley/Couch