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Presented To : Dr. AmberPresented By: Maryam Shakeel
It is a large scale study of proteins, particularly their structures and functions.
Y2H is a molecular biology technique
In vivo technique Protein-protein interaction
Premise
Transcriptional activation
Reporter gene
1. Two Domains of Transcriptional activators
Transcriptional Activator
DNA Binding Domain
Activating Domain
Recognize specific sequence
Enable RNA Polymerase to
transcribe reporter gene
Gal4
X
Y z
X = Bait ProteinY= Pray Protein
Two hybrid proteins are generated with transcription factor domains
Interaction between Bait and Prey is necessary for transcription.
Reporter Gene
X
Binding
Domain
Y
Activation
Domain
UAS
X transcription
The reporter gene use in Gal4 system is LacZgene.
Reporter Gene
X
Binding
Domain
Y
Activation
Domain
UAS
Protein-protein interaction can be measure by measuring transcription of reporter gene.
Bait Plasmid
Prey Plasmid
TRP use as selectable marker
Leu used as selectable marker
Prey Plasmid
Bait Plasmid
Strain carry mutations in different genes for A.A biosynthesis such as TRP1, LEU2, HIS3 and URA3.
Grow in growth medium lack these A.A Only hybrid plasmid carrying transformant
yeast grow.
1. Highly sensitive 2. Elegant mean to study protein-protein
interaction.3. Characterize these interaction
4. A fairly new addition to the family of microbiological studies
Which protein domain is
responsible
Conditions for interaction
Limited to those proteins
Transcription occurs only when DBD bound to AD
Localize in nucleus
Stable in yeast cell
Continue its activity in yeast cell
Protein-Protein Interactions: Methods and Applicationsedited by Haian Fu
The Yeast Two-hybrid Systemedited by Paul L. Bartel, Stanley Fields
http://labrat.fieldofscience.com/2010/01/exploring-protein-interactions-yeast.html
http://www.hybrigenics-services.com/contents/resources/yeast-two-hybrid-principle