1. Yeast Two-Hybrid Screening: The Principles
11/6/2018
Yeast Two-Hybrid Screening: The Principles
Dr. Manfred Koegl

2. How the yeast two-hybrid system (Y2H) works
To look at the principles of yeast two hybrid screening, let‘s forget about protein-protein interactions for a minute, and look at a transcription factor: Many transcription factors have these two domains: BD
A DNA-binding domain (BD),
for promoter recognition, AD
and an activation domain, for transcriptional activation.
promoter
gene

3. A split transcription factor
When these two domanis are expressed as seperate proteins, the BD will still bind to DNA, but the AD is not in the right place to activate transcription. AD BD
promoter
gene

4. Hybrid proteins as molecular glue
To test if two proteins (here: X and Y) interact, they are are expressed in fusion with the BD and the AD. AD BD Y X
promoter
gene

5. A reconstituted transcription factor
If proteins X and Y bind to each other, the transcription factor is reconstituted, and gene expression is activated. AD BD X Y
promoter
gene

6. AD BD gene GAL4 GAL4- GAL4- AD BD
In the Y2H screens running in the high-throughput facility at the DKFZ, the reconstituted transcription factor is the yeast GAL4 transcriptional activator.
GAL4- AD AD BD
GAL4- BD
gene
GAL4-UAS
The DNA-binding domain of GAL4 binds to the GAL4-UAS (upstream activating sequence) on DNA.

7. AD BD reporter gene Reporter genes
To monitor transcriptional activation, reporter proteins such as b-galactosidase are expressed under the control of the GAL4-upstream activating sequence. AD BD
reporter gene
GAL4-UAS

8. Summary wild-type GAL4: reporter expression on split GAL4:
(This is just my symbol for a budding yeast cell!) BD AD
wild-type GAL4:
reporter expression on
reporter BD AD
split GAL4:
reporter expression off
reporter BD AD
reconstituted GAL4:
reporter expression on
reporter

9. Reporter gene examples
HIS3
Used in yeast strains lacking the HIS3 gene
Activation  growth in medium lacking histidine
Advantage: enrichment of clones which harbour interacting proteins
MEL1 (a-galactosidase)
Cleavage of fluorogenic a-D-galactopyranosides
Activation  fluorescence signal
Advantage: quantitative readout

10. Y2H Screens: Bait and prey
Typically, in a Y2H screen, novel binding partners are sought for a „bait“ protein, i.e. a protein of interest (protein X on slide 5).
The bait is expressed in fusion with the DNA-binding domain from the GAL4 transcriptional activator (protein Y on slide 5).
To provide the second hybrid protein, a cDNA-library is used that expresses cDNAs in fusion with the transcriptional activation domain of GAL4.

11. with the DNA-binding domain of a transcription factor
Bait and prey plasmids
bait
library (prey)
Yeast
promoter
GAL4-DBD
GAL4-AD
bait plasmid
prey plasmid (library)
These plasmids express fusion proteins of ...
...your favorite gene
with the DNA-binding domain
of a transcription factor
....a cDNA library
with the activation domain
of a transcription factor

12. Yeast two-hybrid screens
A cDNA library:
one bait
many different preys
„positive clone“
Selection based on reporter gene expression
 isolation of yeast cells harbouring a prey cDNA fragment coding for a protein that binds to the bait.

13. Analysis of positive clones
Sequence analysis of the library insert
Selection of reliable interactions by simple statistic criteria, such as
Frequency of occurrence with the specific bait  reproducibilty of the interaction
 include reproducible interactions!
Frequency of occurrence with other baits „stickyness“ of the prey
 exclude sticky preys!
Definition of high confidence datasets
 fraction of false positives 40% or less
See also Albers et al., Molecular and Cellular Proteomics 2005, 4:205-13

14. End