Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Department of Physiology, Biophysics and Systems Biology, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA.
Nucleic Acids Res. 2020 Jan 10;48(1):e6. doi: 10.1093/nar/gkz1066.
Eukaryotic mRNAs are predominantly translated via the cap-dependent pathway. Initiation is a rate-limiting step in cap-dependent translation and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for characterizing the cap-dependent initiation kinetics. Here, we report an in vitro single-molecule assay that allows characterization of both initiation and peptide chain elongation kinetics for cap-dependent translation. Surprisingly, the histogram of the first-round initiation time is highly asymmetrical and spans a large time range that is several-fold greater than the average peptide synthesis time in translation reactions with a firefly luciferase-encoding mRNA. Both the histogram and single-molecule trajectories reveal an unexpected high-degree of asynchrony in translation activity between mRNA molecules. Furthermore, by inserting a small stem-loop (ΔG = -4.8 kcal/mol) in the middle of the mRNA 5' untranslated region (UTR), our assay robustly detects small changes in budding yeast initiation kinetics, which could not be resolved by bulk luminescence kinetics. Lastly, we demonstrate the general applicability of this assay to distinct cell-free translation systems by using extracts prepared from budding yeast, wheat germ, and rabbit reticulocyte lysates. This assay should facilitate mechanistic studies of eukaryotic cap-dependent translation initiation and translational control.
真核生物 mRNA 主要通过帽依赖途径进行翻译。起始是帽依赖翻译中的限速步骤，也是翻译调控机制的主要靶点。目前缺乏用于描述帽依赖起始动力学的高分辨率技术。在这里，我们报告了一种体外单分子测定法，该方法可用于表征帽依赖翻译的起始和肽链延伸动力学。令人惊讶的是，第一轮起始时间的直方图高度不对称，跨越了一个大的时间范围，比萤火虫荧光素酶编码 mRNA 的翻译反应中的平均肽合成时间长几倍。直方图和单分子轨迹都揭示了 mRNA 分子之间翻译活性的出乎意料的高度异步性。此外，通过在 mRNA 5'非翻译区（UTR）的中间插入一个小茎环（ΔG = -4.8 kcal/mol），我们的测定法能够稳健地检测到酵母起始动力学的微小变化，而这些变化无法通过批量发光动力学来解析。最后，我们通过使用从芽殖酵母、小麦胚芽和兔网织红细胞裂解物中制备的提取物，证明了该测定法在不同无细胞翻译系统中的普遍适用性。该测定法应有助于真核生物帽依赖翻译起始和翻译调控的机制研究。
