The document discusses the yeast two-hybrid system, which is a technique used to detect protein-protein interactions in vivo. It involves fusing a bait protein to a DNA-binding domain and a prey protein to an activating domain; if the bait and prey proteins interact, they bring the domains together and activate transcription of a reporter gene. This allows researchers to efficiently study protein interactions, characterize the domains involved, and identify conditions required for interaction. However, the yeast two-hybrid system is limited to proteins that can localize and function properly in the yeast cell.

1. Presented To : Dr. Amber
Presented By: Maryam Shakeel

2.  It is a large scale study of proteins, particularly
their structures and functions.

3.  Y2H is a molecular biology
technique
 In vivo technique
 Protein-protein interaction
Premise
Transcriptional
activation
Reporter gene

4. 1. Two Domains of Transcriptional
activators
Transcriptional
Activator
DNA
Binding
Domain
Activating
Domain
Recognize specific
sequence
Enable RNA
Polymerase to
transcribe reporter
gene

5.  Gal4
X
Y z
X = Bait Protein
Y= Pray Protein

6.  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

7. Protein-protein interaction can be measure by
measuring transcription of reporter gene.
 The reporter gene use in Gal4 system is LacZ
gene.
Reporter Gene
X
Y
Binding
Domain
Activation
Domain
UAS

8.  Bait Plasmid
 TRP use as
selectable marker
 Prey Plasmid
 Leu used as
selectable marker
Bait Plasmid
Prey Plasmid

9.  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.

10. 1. Highly sensitive
2. Elegant mean to study protein-protein
interaction.
3. Characterize these interaction
Which protein
domain is
responsible
Conditions for
interaction
4. A fairly new addition to the family of
microbiological studies

11.  Limited to those proteins
Localize in nucleus
Stable in yeast cell
Continue its activity in yeast cell
 Transcription occurs only when DBD
bound to AD

12.  Protein-Protein Interactions: Methods
and Applications
edited by Haian Fu
 The Yeast Two-hybrid System
edited by Paul L. Bartel, Stanley Fields
 http://labrat.fieldofscience.com/2010/01/e
xploring-protein-interactions-yeast.html
 http://www.hybrigenics-services.
com/contents/resources/yeast-two-
hybrid-principle