van der Feltz Clarisse, Hoskins Aaron A
Department of Biochemistry, 433 Babcock Dr., University of Wisconsin-Madison, Madison, WI 53706, USA.
Department of Biochemistry, 433 Babcock Dr., University of Wisconsin-Madison, Madison, WI 53706, USA.
Methods. 2017 Aug 1;125:45-54. doi: 10.1016/j.ymeth.2017.05.011. Epub 2017 May 18.
The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub-populations of molecules with unique behaviors. When smFRET is combined with single-molecule fluorescence colocalization, conformational dynamics can be further linked to the presence or absence of a given spliceosome component. Here, we provide a description of experimental considerations, approaches, and workflows for smFRET with an emphasis on applications for the splicing machinery.
剪接体是一种极其动态的分子机器，其中进行前体mRNA剪接需要组成以及蛋白质和RNA构象发生显著变化。单分子荧光共振能量转移（smFRET）可用于在特征明确的模型系统和整个剪接体中阐明这些动力学。这类单分子数据提供了有关剪接体成分的新信息，可用于识别具有独特行为的分子亚群。当smFRET与单分子荧光共定位相结合时，构象动力学可以进一步与给定剪接体成分的存在与否联系起来。在这里，我们描述了smFRET的实验考虑因素、方法和工作流程，重点是剪接机制的应用。
