Gaba Anthony, Wang Hongyun, Qu Xiaohui
Molecular Biology Program, Memorial Sloan Kettering Cancer Center.
Molecular Biology Program, Memorial Sloan Kettering Cancer Center;
J Vis Exp. 2020 Sep 15(163). doi: 10.3791/61648.
Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide-ribosome-mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.
帽依赖性蛋白质合成是真核细胞中主要的翻译途径。虽然各种生化和遗传学方法已允许对帽依赖性翻译及其调控进行广泛研究，但该翻译途径的高分辨率动力学特征仍很缺乏。最近，我们开发了一种体外测定法，以单分子分辨率测量帽依赖性翻译动力学。该测定法基于荧光标记抗体与新生的表位标签多肽的结合。通过对抗体与新生肽 - 核糖体 - mRNA复合物的结合和解离进行成像，可以跟踪单个mRNA上的翻译进程。在这里，我们提供了建立该测定法的方案，包括mRNA和聚乙二醇化载玻片的制备、翻译的实时成像以及单分子轨迹分析。该测定法能够跟踪单个帽依赖性翻译事件，并解析关键的翻译动力学，如起始和延伸速率。该测定法可广泛应用于不同的翻译系统，并应广泛有益于帽依赖性翻译动力学和翻译控制机制的体外研究。
