Yeast Two-Hybrid Screening: The Principles 11/6/2018 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: BD A DNA-binding domain (BD), for promoter recognition, AD and an activation domain, for transcriptional activation. promoter gene

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

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

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

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.

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

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

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

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.

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

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.

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

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