7 YEAST GENETICS
23 Two-hybrid analysis is a powerful way to identify protein-protein interactions in vivo
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Another popular approach for detecting interactions is a two-hybrid analysis (952).
This method is designed to detect physical interactions between two proteins in vivo.
Two-hybrid analysis relies on two characteristics of transcriptional activator proteins:
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Many
transcriptional activator proteins contain two functional domains: a
DNA-binding domain and a transcriptional activation domain.
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These
two domains can often be physically separated. That is, if the
DNA-binding domain is present on one protein and the transcriptional
activation domain is present on a second, interacting protein,
interaction of the two proteins will activate transcription.
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Thus, we can make transcriptional activation
dependent on a particular protein-protein interaction in vivo.
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Figure 7.30
Proteins that interact with each other can be
identified by their ability to activate transcription by linking a
DNA-activating domain to a DNA-binding domain.
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On the basis of these properties, we can screen S.
cerevisiae for all proteins that physically interact
in vivo with a protein of interest. This is illustrated in Figure 7.30.
First, recombinant DNA methods are used to create a plasmid encoding a
hybrid gene that expresses the protein of interest fused to a known
DNA-binding domain. The most commonly used DNA-binding domains are both
well characterized: one is from the yeast transcriptional activator
Gal4 and the other from the E. coli repressor LexA.
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A recombinant library is then made in which random S. cerevisiae
DNA fragments are cloned into a vector where they can potentially be
fused to a well-characterized transcriptional activation domain. Often,
the Gal4 transcriptional activation domain is used in this type of
vector.
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Next, an S. cerevisiae strain is
used in which the binding sites for the DNA-binding domain (for
example, Gal4 binding sites) are placed 5' of a reporter gene. To
perform the two-hybrid screen, the plasmid that encodes the DNA-binding
domain fusion protein is first used to transform S. cerevisiae.
Then, the library of fusions to the activation domain is used to
transform the same strain. Individual transformants are screened or
selected (depending on the reporter used) to identify those in which
transcription of the reporter has been activated. Such cotransformants
are candidates to have a two-hybrid interaction. The determination of
the DNA sequence in the activator plasmid identifies the putative
interactor. Subsequent biochemical and genetic tests can confirm a
physical interaction.
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Besides suppressor analysis and
two-hybrid analysis, yeast geneticists employ many other types of
approaches to identify functionally related genes. Which method is the
best one to use? To answer that question requires being able to predict
the future. We never know what genes will be discovered; however, the
long and successful history of these and related approaches holds the
promise of new and exciting insights.
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Last Revised on October 12, 2004
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952 Fields, S. and Song, O.
(1989).
A novel genetic system to detect protein-protein interactions.
Nature 340, 245-246.
PubMed Journal
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©Jones and Bartlett Publishers (2007)
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