Biomedical Engineering Reference
In-Depth Information
(a)
AD
AD
AD
BD
BD
BD
Reporter
(b)
AD
AD
AD
AAD
AD
AAD
BD
BD
Bait
Bait
Prey 1
Prey 2
Prey 3
x
x
x
Reporter
x
x
x
Reporter
Reporter
Figure 8.1
Principles of the classical Y2H system. (a) The Gal4 transcription factor is composed
of two functionally distinct domains: the DNA binding domain (BD), which recognizes specific
sequences in the promoter region of GAL4-dependent genes; and a transcriptional activation
domain (AD), which drives transcription of adjacent downstream genes. In the Y2H system
the Gal4 protein is fragmented into its two component domains. The DB domain is fused to
a protein of interest and the AD domain is fused to a potential binding partner. If the two
proteins interact, the DB and AD domains are brought into close proximity and are capable of
driving transcription of a downstream reporter gene. (b) When potential binding partners of a
protein of interest are not known, bait constructs can be used to screen a library of potential
interaction partners. In this method, yeast containing a specific bait protein is mated against
many thousands of yeasts containing different prey proteins. Only when bait and prey proteins
interact will biosynthetic reporter genes be turned on. This enables yeast containing interacting
proteins to grow on selective media. Sequencing of DNA encoded in the prey vector enables the
identity of novel interaction partners to be identified.
