Biomedical Engineering Reference
In-Depth Information
Figure 2 . Schematic overview of the yeast two-hybrid assay. See text for details. DBD =
DNA binding domain; TAD = transcriptional activation domain.
The assay is carried out in yeast cells that harbor an engineered yeast gene,
called a reporter gene, whose upstream region contains a short DNA sequence to
which the DNA binding domain of the transcription factor can bind. (Reporter
genes are genes whose expression can be easily monitored.) If the two hybrid
proteins are expressed together in the same cell, the reporter genes will be ex-
pressed if and only if proteins A and B physically interact, and thus build a
bridge between the transcriptional activation and the DNA binding domain of
the transcription factor (Figure 2).
The two-hybrid approach has been applied to detect not only interactions
among two proteins but also on a much larger scale to detect interactions of
most proteins in a genome (4,11,12,18,19,28,30). This is a formidable task, con-
sidering that even for a small genome like that of the yeast Saccharomyces cere-
visiae , with some 6,000 genes, 18 million pairwise interactions need to be
screened. One approach in carrying out such massive screens takes advantage of
the fact that yeast cells reproduce facultatively sexually. They come in two hap-
loid mating types called a and B, which can fuse to form a diploid cell.
To screen for all pairwise protein interactions in a genome, one can generate
two libraries of molecules, including a bait library containing fusions of all the
genes encoding proteins of interest with the coding region for the DNA binding
domain of a yeast transcription factor. After yeast cells of mating type a are
transformed with this library, each transformed cell will express a fusion protein
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