protein-protein interaction screens. bacterial two-hybrid system selectable marker rna polymerase...

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Protein-Protein Interaction Screens

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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

Protein-Protein Interaction Screens

Page 2: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Bacterial Two-Hybrid System

selectable markerRNA polymerase

DNA bindingprotein

bait

target sequence

target

Page 3: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Yeast Two-Hybrid System

DNA bindingprotein

activationdomaintranscription

factor

Page 4: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Yeast Two-Hybrid System

transcriptionfactor

activationdomain

DNA bindingprotein

bait

targ

et

Page 5: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Yeast Two-Hybrid System

selectable markerRNA polymerase

transcription factorbinding site

DNA bindingprotein

bait activationdomain

target

Page 6: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

His3 as Selectable Marker and 3-AT

Page 7: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

3-amino triazole (3AT)

NH

NN

NH3

Page 8: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

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

Page 9: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Libraries for 2-Hybrid Screens

Page 10: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Libraries for 2-Hybrid Screens

gene 1 gene 2 gene 3 gene 4 gene 5 gene 6

X X X X X

Page 11: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

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)

Page 12: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Library Construction

target genegenome

PCR primers

activation domain/polymerase subunit

target gene

Page 13: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

cDNA

Library Construction

target gene (coding region)mRNA

primercDNA

cDNA

fragment

Page 14: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Library Construction

activation domain/polymerase subunit

activation domain/polymerase subunit

activation domain/polymerase subunit

Page 15: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Library Construction

target fragment selectable markeractivation domain

Page 16: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Whole-Genome Two-Hybrid Screens

• Construction of Bait and Target Libraries Covering Entire Proteome

• Automated Screening for Positives

Page 17: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Yeast Two-Hybrid Screen

(Uetz et al., Nature 2000)

Page 18: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Page 19: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

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?

Page 20: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

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)

Page 21: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

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

Page 22: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Page 23: Protein-Protein Interaction Screens. Bacterial Two-Hybrid System selectable marker RNA polymerase DNA binding protein bait target sequence target

Other Methods to ID Protein-Protein Interactions

• Co-expression studies

• Structure prediction

• Purification of Complexes and Mass-Spec

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