Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.

1. Experimental technique  Yeast two-hybrid system
to identify Protein-Protein interactions

2. Protein-Protein interactions
 Protein–protein interactions are the
physical contacts of high specificity,
established between two or more
protein molecules.
 Proteins control and mediate many
of the biological activities of cells by
these interactions.
 Information about PPIs improves
our understanding of diseases and
can provide the basis for new
therapeutic approaches.
 Aberrant PPIs are the basis of
multiple aggregation-related
diseases, such as Creutzfeldt–
Jakob, Alzheimer's disease, and
may lead to cancer.

3. Examples of protein-protein interactions
Signal Transduction: activity of cells is regulated
through this way in form of extracellular signals
Transport across cell membrane: a protein may be
carrying another protein
Cell metabolism: In many metabolic pathways
different proteins interact to perform a specific
function.
Muscles contraction: myosin filaments act as
molecular motors and by binding to actin enable
filament sliding.

4. What is
Yeast Two
Hybrid
system?
 Also known as Y2H or Two-hybrid screening
 molecular biology technique
 Two Hybrid system is for Isolating and Studying Protein-Protein
Interactions.
 This system uses a reporter gene for detecting the interaction of
pair of proteins in yeast cell nucleus.
 Y2H assay relies on the expression of a reporter gene (such as
lacZ or GFP), which is activated by the binding of a particular
transcription factor.

5. Why Yeast Two Hybrid system
 The yeast two hybrid system has a clear advantage
over classical biochemical or genetic methods
 It is an in vivo technique that uses the yeast cell as a
living test-tube.
 It bears a greater resemblance to higher eukaryotic
systems than a system based on a bacterial host.
 With regards to classical biochemical approaches,
which can require high quantities of purified proteins
or good quality anti- bodies, the two Hybrid system
has minimal requirements to initiate screening, since
only the cDNA of the gene of interest is needed.

6. How Yeast Two Hybrid
system works?
 The DNA sequence that
encodes target protein, is fused
with the DNA that encodes the
DNA binding domain of gene
activator protein using
recombinant DNA technology.
 Interactions are identified by the
transcription of reporter genes.
 Positives are selected using
differential media.

7. The classical Yeast Two Hybrid system
 The early yeast two-hybrid
systems were based on
the finding that many
eukaryotic transcription
factors have separable
DNA-binding and
transcription activation

8.  The protein of interest, the “bait”,
is fused to a DNA-binding
domain.
 Proteins that bind to bait, the
“fish” or “prey”, are fused to a
transcription activation domain

9.  Proteins that do not bind to the
bait will not activate the
transcription of the reporter
gene
 Any protein that binds to the
bait will activate the
transcription of the reporter
gene

10.  The first step is to construct a bait
plasmid and a library. Each type of
plasmid contains a selectable marker
such as an essential amino acid.

15. Advantages
 Immediate availability of the cloned gene of the interacting protein
 Only a single plasmid construction is required
 Interactions are detected in vivo
 Weak, transient interactions can be detected
 Can accumulate a weak signal over time
 Protein purification not necessary
 No antibodies requires

16. Disadvantages
 False positives are the largest problem with the yeast two-hybrid system. Can be
caused by the ability of bait to induce transcription without interaction with the bait
 Possible incorrect protein folding in yeast
 gene encoding target protein must be available
 failed to detect some know interactions
 Elimination of False Positives
 Sequence Analysis
 Retransformation of both strain with bait plasmid and strain without bait plasmid
 Test for interaction with an unrelated protein as bait

17. Conclusion:
 The predictive power of the interactome model allows us to examine
the organization and coordination of multiple complex cellular
processes and determine how they are organized into pathways.
 The interactome model can be used to predict poorly characterized
proteins into their functional context according to their interacting
partners within a module.
 One-to-many relationship can be used for pathway discovery.