Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada.
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Nucleic Acids Res. 2019 Sep 5;47(15):8301-8317. doi: 10.1093/nar/gkz571.
Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
组装因子为细菌 30S 亚基的成熟过程提供速度和方向性。为了更精确地了解这些蛋白质如何介导 30S 成熟，扩大对缺乏特定成熟因子的细菌菌株中纯化的 30S 组装中间体的研究是很重要的。为了揭示必需蛋白 Era 在 30S 核糖体亚基组装中的作用，我们使用定量质谱、高分辨率冷冻电子显微镜和细胞内足迹分析，分析了在 Era 耗尽的大肠杆菌细胞中积累的组装中间体。我们的综合方法允许观察到正在组装的小亚基，并表明除了平台结构域中的关键螺旋外，所有其他 16S rRNA 结构域都能折叠，即使没有 Era。值得注意的是，成熟的颗粒在等待平台结构域成熟时并没有停滞不前，而是重新选择了它们的折叠途径，从而能够协调跨越多个 rRNA 结构域的其他结构基序的成熟。我们还发现，Era 与成熟 30S 亚基的结合使 44 号螺旋和解码中心不稳定，从而阻止了另一个组装因子 YjeQ 的结合。这项工作确立了 Era 在核糖体组装中的作用，并提出了在维持核糖体动态平衡方面的新作用。
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