Hirst M, Ho C, Sabourin L, Rudnicki M, Penn L, Sadowski I
Department of Biochemistry and Molecular Biology; University of British Columbia Vancouver, BC, Canada V6T 1Z3.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8726-31. doi: 10.1073/pnas.141413598. Epub 2001 Jul 10.
We describe a two-hybrid strategy for detection of interactions with transactivator 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 transcription of GAL4-dependent reporter genes. This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80 in vitro. Furthermore, repression of GAL transcription by TUP1-GAL80 requires SRB10, demonstrating that the TUP1 repression domain, in the context of a two-hybrid interaction, functions by the same mechanism as endogenous TUP1. Using this strategy, we demonstrate interactions between the mammalian basic helix-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 library by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions with transactivator proteins in vivo.
我们描述了一种用于检测与反式激活蛋白相互作用的双杂交策略。这种抑制性反式激活蛋白（RTA）系统利用了酵母通用阻遏物TUP1的N端抑制结构域。当TUP1 - GAL80融合蛋白与GAL4共表达时，可抑制GAL4依赖性报告基因的转录。这种效应需要GAL4的C端30个残基，这在体外与GAL80相互作用时是必需的。此外，TUP1 - GAL80对GAL转录的抑制需要SRB10，这表明在双杂交相互作用的背景下，TUP1抑制结构域的作用机制与内源性TUP1相同。利用这一策略，我们证明了哺乳动物碱性螺旋-环-螺旋蛋白MyoD和E12之间以及c-Myc和Bin-1之间的相互作用。我们还通过使用GAL4-VP16作为诱饵融合蛋白，从TUP1 - cDNA融合表达文库中鉴定出了相互作用的克隆。这些结果表明，RTA通常适用于在体内鉴定和表征与反式激活蛋白的相互作用。
