Alternative Yeast Two-Hybrid Systems: The Interaction Trap and Interaction Mating

Since the original demonstration that for proteins P1 and P2 known to interact, it was possible to detect interactions between a DNA-binding domain (DBD) fused P1 and activation-domain (AD) fused P2 by assaying transcriptional activation of one or more reporter genes containing cognate DNA binding site for the DBD (), a number of groups have developed variants of this approach for purposes such as mapping interaction domains on proteins, screening cDNA libraries for novel interacting proteins, and screening novel proteins against predetermined sets of known proteins to identify which pairs interact. We here describe strategies for these purposes utilizing the interaction trap (), a two-hybrid system variant developed in the laboratory of Roger Brent (Fig. 1). Open image in new window Fig. 1. The interaction trap. In the basic application, an EGY48 or EGY191 yeast cell contains two LexA-operator responsive reporters, the first a chromosomally integrated copy of the LEU2 gene (required for growth on media lacking leucine), and the second a plasmid bearing a GAL1 promoter-LacZ fusion gene (causing yeast to turn blue on media containing X-gal). These yeast additionally contain a plasmid constitutively expressing the DNA-binding domain of LexA-fused to the "bait" protein P1: and a plasmid which is induced by galactose to express an activation domain-fused cDNA library or specific protein P2. On glucose medium (A), the DBD-fused protein is unable to activate expression of either of the two reporters, so yeast are unable to grow on medium lacking leucine, and are white on medium containing X-gal. On galactose medium (B), the AD-fusion protein is induced: a positive interaction is shown in which the DBD and AD-fused proteins interact strongly, resulting in activation of the two reporters, thus causing growth on media lacking leucine, and blue color on media containing X-gal.