蛋白质合成的荧光共振能量转移（FRET）研究
Ensemble and single-molecule FRET studies of protein synthesis.
机构信息
Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, United States.
Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, United States.
出版信息
Methods. 2018 Mar 15;137:37-48. doi: 10.1016/j.ymeth.2017.12.007. Epub 2017 Dec 13.
Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges.
蛋白质合成是一个复杂的、多步骤的过程，涉及核糖体和翻译蛋白因子的大构象变化。在过去的十年中，荧光共振能量转移（FRET）已成为研究翻译装置结构重排的重要工具。在这里，我们讨论了用于研究翻译的整体和单分子（sm）FRET 测定的设计。我们描述了许多可用于将荧光团引入核糖体、tRNA、mRNA 和翻译蛋白因子的实验策略。还回顾了 smFRET 实验中将翻译组件固定在显微镜载玻片上的替代方法。最后，我们讨论了 FRET 数据解释中的可能挑战以及解决这些挑战的方法。
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