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Yeast two-hybrid system overview

The yeast two-hybrid system is based on the fact that eukaryotic transcriptional activators consist
of two individual domains, the DNA binding domain (DBD) and the activation domain (AD).

The DBD recognizes a specific DNA sequence. The AD coordinates the assembly of the
elements required for transcription and enables RNA polymerase II to transcribe a specific
reporter gene downstream of the DBD domain.

Using the yeast two-hybrid system the protein of interest (X) is expressed as a fusion protein to
the DBD (DBD-X; also known as the “bait” protein) and the activation domain is fused to the
second protein of interest (Y), (AD-Y; also known as the “prey” protein).

The AD-Y fusion vector is introduced into a yeast strain containing the DBD-X fusion partner by
transformation or mating. Only if proteins X and Y physically interact with one another are the
DBD and AD brought together to activate expression of the downstream reporter gene.

See the diagram.

Each of the genes that code for the two proteins of interest (Pr1 and Pr2 in the figure shown) is
fused to a transcription factor and then the pair of hybrid gene is expressed in yeast.
The transcription-factor component ( a DNA-binding factor (DBD) and an activation domain
AD) encoded by the two different hybrids will activate a reporter gene in the yeast , but only if
they become associated with each other to form an active transcription factor.

This only happens when the two gene products of interest interact with each other to form a
complex. When the hybrid proteins of interest form a complex, the transcription-factor pieces
are also brought together, the reporter gene is activated and a signal is detected.

This assay has done much to help establish protein-protein interactions for proteins from a
variety of species.

Yeast 2 Hybrid Limitations

Important * False positive result.

Purpose: Increasing specificity in high-throughput yeast two-hybrid experiments

After yeast two-hybrid system screening, other methods, such as in vitro binding assays, co-
assays and function analysis of the cloned cDNA, are routinely used to rule out false-positive
clones and verify protein-protein interactions.

Y2H library screening generates a significant number of false-positive interactions.

Two main categories of false-positives can be distinguished.

The first consists of ‘‘biological’’ false-positives: protein–protein interactions that occur in yeast
cells, but do not occur in vivo in the organism of study, because the two proteins are not
expressed at the same time or in the same tissues.

These are difficult to eliminate without extensive biological knowledge of the proteins in

The second category consists of ‘‘technical’’ false-positives: protein–protein interactions that are
identified in Y2H screens due to technical limitations of the system.

Note: Get additional info about false positive.