免疫沉淀蛋白复合物的单分子方法：miRNA 尿苷酸化的新见解。
Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation.
机构信息
School of Biological Sciences and Creative Research Center, Seoul National University, Seoul 151-742, Korea; Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Lorentzweg 1, Delft 2628 CJ, The Netherlands.
出版信息
EMBO Rep. 2011 Jul 1;12(7):690-6. doi: 10.1038/embor.2011.100.
Abstract
Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level.
摘要
由于研究需要辅助因子或翻译后修饰的蛋白质存在困难，单分子技术仅应用于生物问题的一个子集。在这里，我们提出了一种新的方法，将单分子荧光显微镜和免疫沉淀结合起来研究蛋白质复合物。我们使用这种方法来研究 Lin28 介导的 TUT4（末端尿苷转移酶 4，多 U 聚合酶）对 microRNA 的尿苷酸化，这调节 let-7 microRNA 的生物发生。我们对 TUT4 免疫沉淀物的尿苷酸化的实时分析表明，Lin28 作为 TUT4 的持续性因子发挥作用。我们的新技术 SIMPlex（免疫沉淀复合物的单分子方法）为在单分子水平上分析复杂蛋白质提供了一种通用工具。
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