Transcript Document 7452559

5/23/2016
Yeast Two-Hybrid Screening:
The Principles
Dr. Manfred Koegl
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:
A DNA-binding domain (BD),
for promoter recognition,
BD
and an activation domain,
for transcriptional activation.
AD
promoter
5/23/2016 | Page 2
Dr. Manfred Koegl
gene
Genomics and proteomics core facilities
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
5/23/2016 | Page 3
Dr. Manfred Koegl
gene
Genomics and proteomics core facilities
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.
BD
X
Y
promoter
5/23/2016 | Page 4
Dr. Manfred Koegl
AD
gene
Genomics and proteomics core facilities
A reconstituted transcription factor
If proteins X and Y bind to each other, the transcription factor is
reconstituted, and gene expression is activated.
BD
X
Y
AD
promoter
5/23/2016 | Page 5
Dr. Manfred Koegl
gene
Genomics and proteomics core facilities
GAL4
In the Y2H screens running in the high-throughput facility at the DKFZ, the
reconstituted transcription factor is the yeast GAL4 transcriptional
activator.
GAL4BD
BD
GAL4AD
AD
gene
GAL4-UAS
The DNA-binding domain of GAL4
binds to the GAL4-UAS (upstream
activating sequence) on DNA.
5/23/2016 | Page 6
Dr. Manfred Koegl
Genomics and proteomics core facilities
Reporter genes
To monitor transcriptional activation, reporter proteins such as b-galactosidase
are expressed under the control of the GAL4-upstream activating sequence.
BD
AD
reporter gene
GAL4-UAS
5/23/2016 | Page 7
Dr. Manfred Koegl
Genomics and proteomics core facilities
Summary
(This is just my symbol for
a budding yeast cell!)
BD AD
reporter
AD
BD
reporter
BD
AD
reporter
5/23/2016 | Page 8
wild-type GAL4:
reporter expression on
Dr. Manfred Koegl
split GAL4:
reporter expression off
reconstituted GAL4:
reporter expression on
Genomics and proteomics core facilities
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
5/23/2016 | Page 9
Dr. Manfred Koegl
Genomics and proteomics core facilities
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.
5/23/2016 | Page 10
Dr. Manfred Koegl
Genomics and proteomics core facilities
Bait and prey plasmids
bait
Yeast
promoter
library (prey)
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
5/23/2016 | Page 11
Dr. Manfred Koegl
....a cDNA library
with the activation domain
of a transcription factor
Genomics and proteomics core facilities
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.
5/23/2016 | Page 12
Dr. Manfred Koegl
Genomics and proteomics core facilities
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
5/23/2016 | Page 13
Dr. Manfred Koegl
Genomics and proteomics core facilities
End
5/23/2016 | Page 14
Dr. Manfred Koegl
Genomics and proteomics core facilities