It is well recognized that many
cellular processes are performed by protein machines composed of large
protein assemblies. Large-scale efforts to detect and characterize
protein assemblies using yeast two-hybrid technology are under
way. The yeast two-hybrid system has been the method of choice used to
analyze a pair of proteins that physically interact mainly because is
simple and can be scaled to study a large number of
interactions. However, there are a number of reasons why similar
genetic assays in E. coli should be useful, including:
higher transformation efficiencies, lack of endogenous proteins and
the absence of cellular compartments. This work presents a
complementary approach based on finding peptides encoded by random
fragments of yeast DNA that can participate in protein
assemblies. Lambda repressor fusion proteins encoding oligomerization
units reconstitute the activity of the lambda repressor. Cells
expressing active repressor fusions have a distinct phenotype that is
easily detected using reporter genes or phage immunity. A recent
report from the Fields lab describes a list of 957 putative
interactions from proteins encoded in the yeast genome, 37 of those
(3.8%) represent proteins that can self-assemble. Here we show that
the repressor system can provide complementary information by
identifying interacting domains within these proteins and by detecting
self-interactions of that were missed in the yeast two-hybrid
analysis.
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