Abstract：
One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.
摘要：
蛋白质合成的最关键步骤之一是信使RNA（mRNA）和转移RNA（tRNA）的偶联易位，这是将mRNA阅读框提前一个密码子所需的。在真核生物中,易位加速，其保真度由伸长因子2(eEF2)1,2保持。目前,仅报道了一些真核核糖体易位的快照3-5。在这里，我们报告了与mRNA组成的完整易位模块结合的延伸真核核糖体的十种高分辨率低温电子显微镜（cryo-EM）结构，肽基tRNA和脱酰基tRNA，其中七个还含有核糖体结合，自然修饰的eEF2。这项研究概述了mRNA-tRNA2生长的肽模块通过核糖体的进展，从eEF2转位酶适应的最早状态到该过程的最后阶段，并显示了一个复杂的相互作用网络，防止了平移阅读框架的滑动。我们证明了真核易位的准确性如何依赖于80S核糖体的真核生物特异性元件，eEF2和tRNA。我们的发现揭示了抗真菌eEF2结合抑制剂翻译阻滞的机制，sordarin.我们还建议由二苯甲酰胺施加的空间约束环境，eEF2中保守的真核翻译后修饰，不仅可以稳定正确的Watson-Crick密码子-反密码子相互作用，还可以揭示错误的肽基-tRNA，因此有助于提高真核生物蛋白质合成的准确性。
