Wang Yuhong
Department of Biology and Biochemistry, University of Houston;
J Vis Exp. 2021 Jul 6(173). doi: 10.3791/62664.
The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.
核糖体是一种大型核糖核蛋白复合物，它沿着mRNA模板逐步组装蛋白质。核糖体的直径约为20纳米，以便在A、P和E位点容纳大的tRNA底物。因此，核糖体动力学自然会很快失相。单分子方法可以分别检测每个核糖体并区分不均匀群体，这对于揭示多组分系统的复杂机制至关重要。我们报告了一种基于尼康Ti2倒置显微镜的单分子荧光共振能量转移（smFRET）方法的详细信息，以探测核糖体蛋白L27和tRNA之间的核糖体动力学。L27在其独特的半胱氨酸53位置进行标记，并重组到一个经过工程改造而缺乏L27的核糖体中。tRNA在其肘部区域进行标记。在延伸循环过程中，当tRNA移动到核糖体内的不同位置时，例如转位前和转位后，FRET效率和动力学表现出差异，这表明存在多个亚群。这些亚群是整体方法无法检测到的。基于全内反射荧光（TIRF）的smFRET显微镜建立在手动或电动倒置显微镜上，采用自制的激光照明。核糖体样品通过超速离心纯化，加载到自制的多通道样品池中，然后通过倏逝激光场进行照明。反射激光光斑可用于实现完美聚焦的反馈控制。荧光信号由电动滤光片转塔分离，并由两个数字互补金属氧化物半导体（CMOS）相机收集。强度通过NIS-Elements软件获取。
