protein-protein interaction screens. bacterial two-hybrid system selectable marker rna polymerase...
Post on 22-Dec-2015
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![Page 1: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target](https://reader036.vdocument.in/reader036/viewer/2022062407/56649d7a5503460f94a5e226/html5/thumbnails/1.jpg)
Protein-Protein Interaction Screens
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Bacterial Two-Hybrid System
selectable markerRNA polymerase
DNA bindingprotein
bait
target sequence
target
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Yeast Two-Hybrid System
DNA bindingprotein
activationdomaintranscription
factor
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Yeast Two-Hybrid System
transcriptionfactor
activationdomain
DNA bindingprotein
bait
targ
et
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Yeast Two-Hybrid System
selectable markerRNA polymerase
transcription factorbinding site
DNA bindingprotein
bait activationdomain
target
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His3 as Selectable Marker and 3-AT
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3-amino triazole (3AT)
NH
NN
NH3
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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
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Libraries for 2-Hybrid Screens
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Libraries for 2-Hybrid Screens
gene 1 gene 2 gene 3 gene 4 gene 5 gene 6
X X X X X
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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)
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Library Construction
target genegenome
PCR primers
activation domain/polymerase subunit
target gene
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cDNA
Library Construction
target gene (coding region)mRNA
primercDNA
cDNA
fragment
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Library Construction
activation domain/polymerase subunit
activation domain/polymerase subunit
activation domain/polymerase subunit
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Library Construction
target fragment selectable markeractivation domain
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Whole-Genome Two-Hybrid Screens
• Construction of Bait and Target Libraries Covering Entire Proteome
• Automated Screening for Positives
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Yeast Two-Hybrid Screen
(Uetz et al., Nature 2000)
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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?
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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)
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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
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Other Methods to ID Protein-Protein Interactions
• Co-expression studies
• Structure prediction
• Purification of Complexes and Mass-Spec