Protein–protein interactions occur in many biological processes including replication, transcription, secretion, signal transduction, and metabolism. A fundamental question in the study of any protein is to identify proteins that interact with a given protein in vivo. Intense research efforts are focused on the identification of these proteins.
The HybriZAP-2.1 two-hybrid vector system (Figure 1), a eukaryotic system to detect protein–protein interactions in vivo, provides a method for the rapid identification of genes encoding proteins that interact with a given protein (i.e., a bait protein). The system is based on the ability to separate eukaryotic transcriptional activators into two separate domains, the DNA-binding domain (BD) and the transcriptional activation domain (AD). In the HybriZAP-2.1 two-hybrid vector system, proteins that interact with the bait protein are identified by generating hybrids of the yeast GAL4 BD and the bait protein (X) and the GAL4 AD and a library of proteins (Y). Neither hybrid protein is capable of initiating specific transcription of reporter genes in yeast in the absence of a specific interaction with the other hybrid protein (Figure 2A).
When the hybrid protein X is expressed in yeast, the GAL4 BD binds X to specific DNA sequences in the yeast chromosome defined by the GAL1 or GAL4 upstream activating sequences (UASGAL1 or UASGAL4, respectively), which regulate the expression of a reporter gene. Binding of X to the UAS is not sufficient to initiate transcription of the reporter gene. When Y is expressed in yeast, the AD interacts with other components of the transcription machinery required to initiate transcription of the reporter gene. However, Y alone is not localized to the reporter gene UAS and therefore does not activate transcription of the reporter gene. When a specific interaction between X and Y localizes both the GAL4 BD and GAL4 AD to the reporter gene UAS, transcriptional activation of the reporter gene occurs (Figure 2B). The reporter genes in the HybriZAP-2.1 two-hybrid vector system are ß-galactosidase (lacZ) and histidine (HIS3).
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