In Lecture 1 we learned about two methods for identifying protein interactions: Yeast Two-Hybrid and CoImmunoprecipitation. These techniques are designed to identify physical bindings between proteins. 1.1.1 Yeast Two-Hybrid (Y2H) The yeast two-hybrid technique [17, 9] allows the detection pair-wise protein interactions. It exploits the modular property typical of many eukaryotic transcription factors, which can be usually decomposed in two distinct modules, one directly binding to DNA (DB, DNA-binding domain) and the other activating transcription (AD, transcriptional activating domain). The first component, DB, is able to bind to DNA even by itself, while the second module, AD, will activate transcription only if physically associated to a binding domain. In the two-hybrid experiment the test proteins are expressed as fusion proteins (hybrids) with a DNA-binding domain (DB, the bait) and a transcriptional activating domain (AD, the prey). Fusions partners are coexpressed in yeast nucleus where a protein-protein interaction is identified thanks to the activation of the reporter gene, which can be detected and measured. Figure 1 shows that the two proteins whose interaction is under scrutiny, here indicated as bait and prey, are expressed as fusion proteins, respectively, with a binding domain (BD) and an activation domain (AD). If an interaction between bait and prey takes place, the complex formed activates the transcription of the reporter gene, allowing, as a consequence, the detection of the interaction itself.