Yeast two hybrid system / protein-protein interaction

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