Department of Chemistry, KAIST, Daejeon 305-701, Korea.
BMB Rep. 2010 Feb;43(2):110-4. doi: 10.5483/bmbrep.2010.43.2.110.
The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.
酵母三杂交系统（Y3H）是一种强大的鉴定 RNA 结合蛋白的方法，但由于 RNA 非依赖性相互作用，仍存在许多假阳性。在这项研究中，我们试图通过在 RNA 杂交表达载体的 RPR1 启动子中引入四环素操纵子（tetO）基序来有效地识别假阳性。我们成功开发了一种紧密的四环素调控型 RPR1 启动子变体，在转录起始位点和 RPR1 启动子的 A 框序列之间仅含有一个 tetO 基序。在四环素反应性转录激活剂（tTA）存在的情况下，来自该四环素调控型 RPR1 启动子的表达受到四环素衍生物多西环素（Dox）的正向调控。这种开-关控制与多西环素负调控 tTA 的一般知识相反。因此，通过直接监测 RNA 杂交表达，这种正向调控的 RPR1 启动子系统可以有效地消除 Y3H 系统中常见的 RNA 非依赖性假阳性。
