The document discusses yeast hybrid systems, specifically yeast one-hybrid (Y1H), yeast two-hybrid (Y2H), and yeast three-hybrid (Y3H) assays used to study protein-DNA and RNA-protein interactions. Y1H and Y2H differ in that Y1H measures protein-DNA interactions while Y2H measures protein-protein interactions, with Y3H allowing for RNA-protein interaction analysis. The advantages and disadvantages of each system are outlined, highlighting key factors such as sensitivity, specificity, and potential false positives.

1. YEAST HYBRID SYSTEMS

2. Y1H relies on the general principles of the yeast two-hybrid assay. In short,
mammalian proteins are exogenously expressed in yeast and their interactions in
vivo are measured by the downstream activation of reporter gene constructs.
The main difference between Y1H and Y2H is in the interactions that are being
measured – put simply, Y2H measures protein-protein interactions, while Y1H
measures protein-DNA interactions.

3.  In Y2H experiments, the close physical interaction between two proteins (the bait and
the prey) brings together the DNA-binding and activation domains of a transcription
factor. This forms a functional transcriptional unit that turns on expression of a reporter
gene.
 In a Y1H system, detection is based on the interaction of a single protein (the prey) with
a bait DNA sequence positioned upstream of a reporter gene. Importantly, the prey
protein is fused to a transcriptional activation domain. Positive protein-DNA interactions
bring the fusion activation domain into close proximity with the promoter element, thus
activating downstream transcription of the reporter gene

5. 1) A DNA bait sequence. This can be a defined promoter sequence, another defined cis-acting sequence, regulatory sequences
from exons, introns, 5’ or 3’-noncoding regions, or even centromere or telomere sequences.
2) Bait plasmid. This also doubles as your reporter plasmid. Unlike Y2H, which uses a standard reporter construct for all bait:prey
interactions, Y1H reporter constructs are specially designed for your experiment to contain the reporter gene (commonly HIS3, a
histidine biosynthesis gene, or LacZ, which encodes for bacterial enzyme β-galactosidase) under the control of a non-endogenous
promoter which lies immediately downstream of your DNA bait sequence. This construct is commonly delivered using a special
plasmid that is able to reliably and stably integrate the sequence by site-specific mutagenesis into a nonessential part of the yeast
genome (e.g. pINT). Successful transformation is then tested using histidine-deficient selection media.
3) Prey plasmid (or plasmids). Much like in a Y2H experiment, your prey plasmid will encode for a protein-of-interest, called the
“prey”, as well as a selection marker, e.g. a leucine biosynthesis gene, which will allow the yeast to grow in medium lacking leucine.
However, a big difference in Y1H is that your prey protein is expressed as a fusion protein with a strong constitutive trans-
activation domain (e.g. Gal4, LexA or VP16) that is able to bind and activate the promoter region just upstream of the reporter
gene. This is to ensure you achieve robust transcriptional activation of the HIS3 reporter gene, regardless of whether the prey
protein has any strong intrinsic activation function.

6. Y1H assay has some clear advantages:
 Unlike luciferase reporter assays, Y1H is able to detect protein-DNA interactions that are
not actually activating transcription. Fusing your protein(s) of interest to a strong
activation domain allows Y1H to detect a variety of DNA-binding proteins, including
those that do not directly function in transcription, e.g. replication proteins, DNA repair
proteins, and repressor proteins.
 Y1H is compatible with many existing libraries. Most Y1H experiments can use hybrid
prey libraries that have been constructed for Y2H applications, e.g. Gal4p- or LexA-
based protein libraries can also be used for screening against various DNA baits in Y1H.
 Y1H can detect isoform-specific interactions. You can design your prey plasmids to
encode specific protein isoforms, allowing detection of highly specific interactions. In
contrast, ChIP-based approaches rely on sensitive and specific antibody binding to your
protein of interest, and these are rarely able to be achieved on an isoform-specific level.

7. Y1H has some disadvantages too:
 Y1H doesn’t provide any information about the functional consequences of a DNA-
protein interaction.
 The rates of false-positive results are high, as either endogenous yeast transcription
factors or prey proteins that can bind Gal4 promoters can activate transcription of the
reporter gene without interacting with the bait DNA sequence.
 Expression of your prey protein fused to an activation domain can result in improper
protein folding, resulting in steric hindrance and loss of interactions, and/or failure to
correctly localise to the nucleus.

8. YEAST 2 HYBRID SYSTEM

9. The yeast-2-hybrid system is a simple scientific technique used to screen
a library of proteins for potential interactions
 Firstly, a transcription factor is broken into two parts – a DNA-binding
domain (BD) and a catalytic activation domain (AD)
 The DNA-binding domain is fused to a protein of interest called the
bait (e.g. an enzyme)
 The activation domain is fused to a number of potential binding
partners – called the prey (e.g. different ligands)
 If the bait and prey interact, the two parts of the transcription factor
are reconstituted and activate transcription of a gene
 If the bait and prey do not interact, the two parts of the transcription
factor remain separate and transcription doesn’t occur

10. The yeast-2-hybrid system detects protein-protein interactions according to the activation of a reporter
gene
The reporter gene may encode for the production of a protein that causes a visible colour change (e.g.
ß-galactosidase)
Alternatively, the reporter gene may encode for the production of an essential amino acid that is required
for the yeast to grow on a deficient media (hence yeast growth would indicate successful interaction
between bait and prey)
Yeast-2-hybrid screens are a simple technique and hence have a relatively high rate of false positives
(partial interactions)
Consequently, the yeast-2-hybrid system is typically only used as an initial test to identify possible
protein interactions

12. YEAST 3 HYBRID ASSAY

13. The three-hybrid system enables the detection of RNA-protein interactions in yeast using simple
phenotypic assays. It was developed in collaboration with Stan Fields laboratory (University of Washington-
Seattle).
In 1996, the Wickens and Kuhl labs developed the yeast three-hybrid system independently. This system
also makes it possible to identify those regions of an RNA or protein that are required for a known
interaction and to test the combinations of RNA and protein to confirm whether they interact in vivo.

14. The Functions of Y3H
It is well-known that Y3H methods can be applied in a variety of research fields:
a. Finding protein partners of a known RNA sequence from cDNA libraries
b. Searching a natural RNA partner or ligand for a known RNA binding protein via RNA libraries
c. Testing suspected RNA-protein interactors
d. Mutational analysis of interacting RNA and protein
e. Discovery of multiprotein-RNA complexes
In addition, Y3H is still a sensitive method to screen for the interactions between small molecule drugs
and their protein targets.

15. The Principle of Y3H
Yeast three-hybrid system is a derivative of yeast two-hybrid (Y2H). It’s a kind of powerful tool to
dissert RNA-protein interactions of interest and that typically consists of three chimeric
components.
The first hybrid protein is made up of an RNA binding protein (RBD) fused to a DNA binding
domain (DBD). The second fusion protein molecule contains a second RNA binding protein fused to
the transcriptional activation domain (AD). The third hybrid part is an RNA molecule which bridges
above two fusion proteins by providing two specific RNA targets for the RNA binding proteins.
When this tripartite constituent forms at a promoter, the reporter gene is turned on, even
transiently. And the expressed reporter products can be recognized by simple biochemical or
phenotypic assays.

17. Key Benefits of Y3H
In an RNA Y3H method, there are lots of great advantages presented on Creative Biolabs’ platforms:
• Fast and sensitive approach to identify and characterize RNA-protein interaction
• Applicable to a wide range of RNA-protein interactions
• Detection of both very strong and very weak interactions of RNA and proteins
• Assay results monitored easily by cell growth, colony color, or the levels of a specific enzyme
• The species of yeast is an ideal model organism in eukaryotic genetics
• Technique improved to reduce the selection of false-positive clones