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292 Serebriiskii et al.for eukaryotic proteins that are not amenable to analysis in the yeast-based system(e.g., DNA-binding domain [DBD]-fused proteins that autoactivate transcription,proteins toxic to yeast, or proteins that have undesired interactionswith endogenous yeast proteins). In contrast, some advantages are specific to theyeast-based system, including the fact that proteins from eukaryotic organismsare more likely to be properly folded and posttranslationally modified in aneukaryotic milieu.Together, the yeast and bacterial two-hybrid systems provide powerfulmethods for analyzing protein–protein interactions. Recently, the creation andoptimization of novel vectors was described that could be used to express DBDfused“baits” that could be used for library screening in either bacterial or yeastenvironments (8). A specific advantage of this system is that it reduces baitcloning and characterization work, facilitating screening for interacting proteinsin yeast and bacterial systems in parallel, and allowing extremely direct comparisonof results obtained in the two systems. As is shown, a single bait used forlibrary screens in yeast and in bacteria could identify very different sets of interactingpartners in the two environments (probably because of the considerationsdiscussed earlier) (8). Hence, use of both systems is more likely to identify afull set of interactive partners for a given bait. This chapter describes the proceduresfor use of these reagents for screening in bacteria; Chapter 15 describesthe use of related reagents (including a common pGLS23 plasmid series) forscreening in yeast.1.1. A Bacterial Two-Hybrid System Basedon Transcriptional ActivationThe bacterial two-hybrid system described herein is based on the observationthat two interacting proteins, “X” and “Y,” can trigger transcriptional activationof a weak promoter in Escherichia coli. As shown in Fig. 1, transcriptional activationis dependent on the expression of two fusion proteins in the cell. One ofthe fusions (the bait) consists of protein X covalently linked to a DNA-bindingprotein, which in turn binds to a specific DNA site positioned near the weakpromoter. In the system described in this chapter, the DNA-binding proteinused for sequence-specific binding comes from bacteriophage λcI repressor.The other fusion (the prey) consists of protein Y (or the proteins encoded by acDNA library) linked to the α-subunit of the E. coli RNA polymerase (RNAP).In this configuration, X is tethered near the weak promoter (through the DNAbindingprotein part of the fusion) and, if X interacts with Y, this recruits RNAPto the weak promoter to thereby activate transcription.In theory, any interacting protein–protein (X–Y) pair should be able to mediatethis transcriptional activation, and a number of experiments have demonstratedthat this is generally true. Interacting protein–protein pairs from prokaryotes