The Yeast Two Hybrid

System

The technique was pioneered by Stanley Fields and Ok-kyu Song in 1989.

It is an important technique used to study protein-protein interactions.

This procedure is based on the discovery that gene expression in S. cerevisiae depends on interactions between pairs of transcription factors or proteins.

A protein is composed of domains, which

allow the same protein to perform different

functions.

The Y2H system uses two protein domains

that have specific functions.

A DNA binding domain(BD) capable of

binding to DNA

An activation domain(AD) capable of

activating transcription of DNA.

The 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.

The query protein of interest fused with the

BD is known as the Bait, and the protein

library fused with the AD is referred to as

the Prey.

The successful interaction between the

proteins is linked to a change in the cell

phenotype.

In the absence of Bait-Prey interaction, the

AD domain is unable to localize to the

reporter gene to drive gene expression.

However, when Bait and Prey interact, the

BD domain binds to the DNA localizing the

AD domain upstream of the reporter gene,

leading to the expression of reporter gene.

.

RECOVERY OF

INFORMATION:

Once the selection has been performed, the

primary structure of the proteins which

display the appropriate characteristics must

be determined. This is achieved by retrieval

of the protein-encoding sequences (as

originally inserted) from the cells showing the

appropriate phenotype.

Advantages of Y2H

system:-

1. Its low tech, can be easily carried out in a

lab.

2. It helps in identification of interaction

partners.

3. Determination of crucial sequences for

interaction.

4.Determination of unknown protein function.

Limitations of Y2H system:-

1. A particular interaction may be absent in

yeast(like if a bacterial protein is tested, the

system may lack the chaperones important

for folding or interaction).

2. If test protein are not localized to the

nucleus, interacting proteins may show a

negative result.

3. Fusion proteins may inhibit the original

interaction.

Chromatin

Immunoprecipitation(ChI

P)

It is a type of immunoprecipitation

experimental technique used to

investigate the interaction between

proteins and DNA in the cell. It aims to

determine whether specific proteins are

associated with specific genomic regions,

such as transcription factors on promoters

or other DNA binding sites.

Procedure:

1) DNA and associated proteins on chromatin

in living cells or tissues are cross linked.

2) The DNA-protein complexes are then

sheared into ~500 bp DNA fragments by

sonication or nuclease digestion.

3) Cross-linked DNA fragments associated

with the protein(s) of interest are selectively

immunoprecipitated from the cell debris using

an appropriate protein-specific antibody.

4)The associated DNA fragments

are purified and their sequence is

determined.

5)Enrichment of specific DNA

sequences represents regions on

the genome that the protein of

interest is associated with in vivo.

.

Types of ChIP :-

1. Cross-linked ChIP

Cross-linked ChIP is mainly suited for mapping the

DNA target of transcription factors or other

chromatin-associated proteins, and uses reversibly

cross-linked chromatin as starting material.

2. Native ChIPNative ChIP is mainly suited for mapping the

DNA target of histone modifiers. Generally, native

chromatin is used as starting chromatin.

ChIP Limitations:

1)It cannot distinguish between transcription

factor isoforms.

2)Large scale assays are challenging, as

antibodies for each TF needs to be made.

3) ChIP does not distinguish between a single

protein and a complex.

4) The results from ChIP cannot be viewed or

analyzed directly, so further protocols such as

Polymerase Chain Reaction (PCR).