Yeast Two-Hybrid Screening: The Principles

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Yeast Two-Hybrid Screening The Principles

• Dr. Manfred Koegl

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How the yeast two-hybrid system (Y2H) works
To look at the principles of yeast two hybrid
screening, lets forget about protein-protein
interactions for a minute, and look at a
transcription factorMany transcription factors
have these two domains
promoter
gene
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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
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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
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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
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GAL4
In the Y2H screens running in the high-throughput
facility at the DKFZ, the reconstituted
transcription factor is the yeast GAL4
transcriptional activator.
AD
GAL4- AD
BD
GAL4- BD
gene
GAL4-UAS
The DNA-binding domain of GAL4 binds to the
GAL4-UAS (upstream activating sequence) on DNA.
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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
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Summary
(This is just my symbol for a budding yeast
cell!)
AD
BD
wild-type GAL4 reporter expression on
reporter
AD
BD
split GAL4 reporter expression off
reporter
AD
BD
reconstituted GAL4 reporter expression on
reporter
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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

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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.

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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
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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.
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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, 4205-13
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End