Assembly of the eukaryotic large ribosomal subunit

项目摘要 核糖体是由核糖体RNA和多达80个核糖体蛋白组成的分子机器。 这些大的集合体催化所有细胞中的蛋白质合成。这个项目的长期目标是 了解真核生物核糖体是如何在200多个非核糖体的帮助下组装的 这些因素是组装中间体的一系列分子快照。结合了遗传、生化 而使用冷冻EM的质谱学方法是工程、捕获、分离和 测定核糖体瞬时组装中间体的原子分辨分子快照 亚单位。 真核细胞核糖体组装可分为四个阶段，共转录组装事件 核仁、核仁中大小核糖体亚基前体的初始成熟 40s前和60s前颗粒的成熟、核输出和细胞质成熟。虽然迟到了 真核细胞核糖体组装中的事件相对较好地描述了早期的共转录 核糖体亚基在核仁中的组装仍然知之甚少。这些非常重要的中间商 早期阶段是极其短暂和短暂的，关键蛋白质复合体的结构 到目前为止，这些早期事件的催化剂仍然难以捉摸。 我的实验室已经开发出新的遗传和生化方法，现在使我们能够有效地 标记、捕获和分离大核糖体亚基的早期核仁组装中间产物。这个 这些方法的协同使用使我们能够克服以前难以处理的生化问题 障碍，从而使详细研究重要的早期组装中间体成为可能 核糖体亚基及其功能之一--多肽出口通道的逐步形成 中锋。这些研究的洞察力将揭示这两种机制在 真核核糖体组装形成功能中心以及真核核糖体中的缺陷 组装可导致人类血液疾病，统称为核糖病。

Project Summary Ribosomes are molecular machines composed of ribosomal RNAs and up to 80 ribosomal proteins. These large assemblies catalyze protein synthesis in all cells. The long-term goal of this project is to understand how eukaryotic ribosomes are assembled with the help of more than 200 non-ribosomal factors as a series of molecular snapshots of assembly intermediates. Combining genetic, biochemical and mass spectrometry approaches with cryo-EM is an essential step to engineer, trap, isolate and determine atomic-resolution molecular snapshots of transient assembly intermediates of ribosomal subunits. Eukaryotic ribosome assembly can be subdivided into four stages, co-transcriptional assembly events and initial maturation of small and large ribosomal subunit precursors in the nucleolus, nuclear maturation of pre-40S and pre-60S particles, nuclear export, and cytoplasmic maturation. While late events in eukaryotic ribosome assembly are relatively well characterized, the early co-transcriptional assembly of ribosomal subunits in the nucleolus is still poorly understood. Intermediates at these very early stages are extremely short-lived and transient and structures of key protein complexes that catalyze these early events have so far remained elusive. My laboratory has developed new genetic and biochemical approaches that now enable us to efficiently tag, trap and isolate early nucleolar assembly intermediates of the large ribosomal subunit. The synergistic use of these approaches has allowed us to overcome previously intractable biochemical hurdles, thereby enabling the detailed study of essential early assembly intermediates of the large ribosomal subunit and the stepwise formation of the polypeptide exit tunnel, one of its functional centers. Insights from these studies will shed light onto both the mechanisms that are employed during eukaryotic ribosome assembly to form functional centers as well as how defects in eukaryotic ribosome assembly can result in human blood disorders, which are collectively termed ribosomopathies.