Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins 1 , 2 . Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1 3 . Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1 4 . The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae . Studies of five cryo-electron microscopy structures reveal the composition and conformational progression in the final maturation events of human 40S ribosomal subunit assembly.

中文翻译：

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人类 40S 核糖体成熟最后步骤的结构基础

真核核糖体由一个小的 40S 亚基和一个大的 60S 亚基组成，它们以高度协调的方式组装。200 多个因素确保核糖体 RNA 的正确修饰、加工和折叠以及核糖体蛋白 1, 2 的及时掺入。当最终的 rRNA 前体 18S-E 在位点 3 被核酸内切酶 NOB1 3 切割时，小亚基成熟在细胞质中结束。人类 40S 前体的先前结构表明，NOB1 由其伙伴 PNO1 4 保持在非活动状态。然而，最终的成熟事件，包括在决定性的 rRNA 切割步骤中激活 NOB1 以及驱动最后生物发生因子解离的机制，仍未得到解决。在这里，我们报告了人类 40S 亚基前体的五种低温电子显微镜结构，它描述了 40S 组装最后步骤中的组成和构象进展。我们的结构解释了 RIOK1 在 PNO1 的置换和解离中的核心作用，这反过来又允许内切核酸酶 NOB1 的构象变化和激活。此外，我们观察到两个因素，真核翻译起始因子 1A 含结构域蛋白 (EIF1AD) 和富含亮氨酸重复序列的蛋白 47 (LRRC47)，它们与 RIOK1 和中央 rRNA 螺旋 44 附近的晚期 pre-40S 颗粒结合。最后，功能数据显示 EIF1AD 是高效组装因子回收和 18S-E 处理所必需的。因此，我们的结果能够详细了解人体细胞中 40S 形成的最后步骤，此外，为人类和模式生物酿酒酵母之间小核糖体亚基形成的主要差异提供证据。对五种冷冻电子显微镜结构的研究揭示了人类 40S 核糖体亚基组装最终成熟事件中的组成和构象进展。