A two-hybrid system for transactivator bait proteins

We describe a two-hybrid strategy for detection of interactions with transa ctivator proteins. This repressed transactivator (RTA) system employs the N -terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when coexpressed with GAL4, are shown to inhibit transcri ption of GAL4-dependent reporter genes, This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80 in vitr o. Furthermore, repression of GAL transcription by TUP1-GAL80 requires SRB1 0, demonstrating that the TUP1 repression domain, in the context of a two-h ybrid interaction, functions by the same mechanism as endogenous TUP1. Usin g this strategy, we demonstrate interactions between the mammalian basic he lix-loop-helix proteins MyoD and E12, and between c-Myc and Bin-1. We have also identified interacting clones from a TUP1-cDNA fusion expression libra ry by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions wit h transactivator proteins in vivo.