Yeast Two Hybrid system uses a
reporter gene to detect the interaction of pair of proteins inside the yeast
cell nucleus. In the yeast Two Hybrid System, The interaction of target protein
to the protein will bring together transcriptional activator, which then
switches on the expression of reporter gene.
reporter gene to detect the interaction of pair of proteins inside the yeast
cell nucleus. In the yeast Two Hybrid System, The interaction of target protein
to the protein will bring together transcriptional activator, which then
switches on the expression of reporter gene.
The Yeast Two Hybrid system makes
use of modular nature of gene activator proteins. These proteins bind to the
DNA and activate the transcription of the gene.
use of modular nature of gene activator proteins. These proteins bind to the
DNA and activate the transcription of the gene.
How the
Yeast Two Hybrid System Works
Yeast Two Hybrid System Works
Even though it looks complex,
Yeast Two Hybrid System is relatively simple to use in labs to study
protein-protein interaction. As the protein-protein interaction occurs inside
the yeast cell nucleus, proteins from every part of the cell and from any
organism can be studied using yeast two hybrid system.
Yeast Two Hybrid System is relatively simple to use in labs to study
protein-protein interaction. As the protein-protein interaction occurs inside
the yeast cell nucleus, proteins from every part of the cell and from any
organism can be studied using yeast two hybrid system.
This is how the Yeast Two Hybrid
System works:
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.
target protein is fused with the DNA that encodes the DNA-binding domain of
gene activator protein using recombinant DNA technology.
Two sets of proteins are used:
·
Bait and the
Prey
Bait and the
Prey
Bait
Target protein is fused to a
DNA-binding domain that localizes it to the regulatory region of a reporter gene as “bait.”
DNA-binding domain that localizes it to the regulatory region of a reporter gene as “bait.”
Prey
Specially designed protein in the cell nucleus (“prey”).
Bait can bind to the regulatory region of the reporter gene,
which acts as a bait and can be used to fish out the protein that interact with
the target protein inside yeast cell. The interaction of Bait and Prey will
bring about the activation and expression / transcription of reporter gene. The
reporter gene is the one which will help the yeast cell grow on selective
medium.
which acts as a bait and can be used to fish out the protein that interact with
the target protein inside yeast cell. The interaction of Bait and Prey will
bring about the activation and expression / transcription of reporter gene. The
reporter gene is the one which will help the yeast cell grow on selective
medium.
Many potential binding partners
can be prepared by ligating DNA encoding the activation domain of gene activator
protein to a large mixture of DNA fragments from a cDNA library.
can be prepared by ligating DNA encoding the activation domain of gene activator
protein to a large mixture of DNA fragments from a cDNA library.
Cells that express this reporter
are selected and grown, and the gene (or gene
are selected and grown, and the gene (or gene
fragment) encoding the prey
protein is retrieved and identified through
protein is retrieved and identified through
nucleotide sequencing.
Through Two Hybrid System a
protein linkage map has been generated for most of the 6,000 proteins in yeast and
similar projects are underway to catalogue the protein interactions in C.
elegans and Drosophila.
protein linkage map has been generated for most of the 6,000 proteins in yeast and
similar projects are underway to catalogue the protein interactions in C.
elegans and Drosophila.
A similar technique called
reverse two hybrid system, which can be used to detect mutation of chemical
compounds that are able to disrupt specific protein interactions. In reverse
two hybrid system the reporter gene can be replaced with a gene that kills the
cells when the bait and prey proteins interact. Eliminating a particular
molecular interaction can reveal something about the role of the participating
proteins in the cell. In addition, compounds that selectively interrupt protein
interactions can be medically useful: a drug that prevents a virus from binding
to its receptor protein on human cells could help people to avoid infections.
reverse two hybrid system, which can be used to detect mutation of chemical
compounds that are able to disrupt specific protein interactions. In reverse
two hybrid system the reporter gene can be replaced with a gene that kills the
cells when the bait and prey proteins interact. Eliminating a particular
molecular interaction can reveal something about the role of the participating
proteins in the cell. In addition, compounds that selectively interrupt protein
interactions can be medically useful: a drug that prevents a virus from binding
to its receptor protein on human cells could help people to avoid infections.
References
Alberts, Molecular Biology of the Cell
Cooper, Molecular Biology of the Cell
The Yeast Two-hybrid System – Paul L. Bartel, Stanley Fields
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