Time-Resolved Visualization of the Yeast Ribosome-Biogenesis by Cryo-Electron Tomography

通过冷冻电子断层扫描对酵母核糖体生物发生进行时间分辨可视化

• 批准号: 400861327
• 负责人: Professor Dr. Achilleas Frangakis
• 金额: --
• 依托单位: Institut für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/400861327?language=en
• 关键词: Time; Resolved; Visualization; Yeast; Ribosome

In this project, we would like to analyze the origins of yeast ribosome biogenesis using cryo-electron tomography (cryo-ET) and correlative light and electron microscopy (CLEM). The ribosome plays a central role in gene expression. It translates the genetic information that is transcribed within the messenger RNA (mRNA) to the proteome of the cell. The importance of the ribosome and the complexity of its structure necessitate a well-coordinated and regulated assembly. Malfunctions of this process are mostly lethal for the cell and can cause severe pathology.While the mature ribosome is well studied, the structural understanding of its biogenesis remains elusive. Eukaryotic ribosome biogenesis starts in the nucleolus with the synthesis of a precursor of the ribosomal RNA. A plethora of ribosome-assembly factors associate with this RNA precursor forming pre-ribosomal particles. We recently visualized these pre-ribosomal particles in the context of the so-called Miller trees (Neyer et al., Nature 2016). Miller trees are supramolecular structures with a ribosomal DNA (rDNA) scaffold that contain actively transcribing RNA polymerase I (Pol I) enzymes, from which the nascent ribosomal RNA (rRNA) chain is emerging and folds to form the pre-ribosomal particles. These pre-ribosomal particles are located at the end of the branches of the Miller trees and are referred to as terminal knobs. Thus, the terminal knobs of the Miller trees visualize the stepwise generation of pre-ribosomes in a quasi-sequential manner. With this application, we would like to analyze the terminal knobs that form co-transcriptionally at the 5’-end of the emerging rRNA through the stepwise addition of protein complexes. Such an analysis approach is only feasible due to the defined localization of the co-transcriptionally assembling complexes on the growing pre-rRNAs of the Miller trees. In yeast, the formation of the terminal knobs is a two-stage process: Early formed knobs will be cleaved from the nascent rRNA chain to form the small ribosomal subunit and are therefore called small subunit (SSU) processome. Later formed knobs will develop to the large ribosomal subunit and are therefore called large subunit (LSU) processome. Thus, the knob generation is a two-stage process that represents the earliest precursors of the final ribosomal subunits. Due to their size (which is approximately 6 MDa for the SSU processome), cryo-electron tomography is ideally suited for the analysis of the terminal knobs. Further on specific labeling of defined states can precisely localize distinct states and facilitate our analysis. Ultimately, this study should result in a more detailed understanding of the structure and function of the SSU and LSU processomes, and provide unprecedented insights into the fascinating mechanism of the early ribosome biogenesis in vivo.

在本项目中，我们将利用冷冻电子断层扫描（cryo-ET）和相关的光学和电子显微镜（CLEM）分析酵母核糖体生物发生的起源。核糖体在基因表达中起着核心作用。它将信使RNA （mRNA）内转录的遗传信息翻译成细胞的蛋白质组。核糖体的重要性及其结构的复杂性需要一个良好协调和调节的组装。这一过程的故障对细胞来说大多是致命的，并可能导致严重的病理。虽然成熟核糖体已经得到了很好的研究，但对其生物发生的结构理解仍然难以捉摸。真核核糖体的生物发生始于核核体RNA前体的合成。过多的核糖体组装因子与这种RNA前体相关，形成核糖体前颗粒。我们最近在所谓的米勒树的背景下可视化了这些核糖体前颗粒（Neyer等人，Nature 2016）。米勒树是一种具有核糖体DNA （rDNA）支架的超分子结构，其中含有活性转录的RNA聚合酶I （Pol I）酶，新生核糖体RNA （rRNA）链从中出现并折叠形成核糖体前颗粒。这些前核糖体颗粒位于米勒树分枝的末端，被称为末端旋钮。因此，米勒树的末端旋钮以准顺序的方式可视化前核糖体的逐步产生。有了这个应用程序，我们想分析通过逐步添加蛋白质复合物在新兴rRNA的5 '端形成共转录的末端旋钮。这种分析方法是可行的，因为Miller树生长的前rnas上的共转录组装复合物的定位是明确的。在酵母中，末端旋钮的形成是一个两阶段的过程：早期形成的旋钮将从新生的rRNA链上切割出来，形成小核糖体亚基，因此被称为小亚基（SSU）过程体。后来形成的旋钮将发展成大核糖体亚基，因此被称为大亚基（LSU）过程。因此，旋钮的产生是一个两阶段的过程，代表了最终核糖体亚基的最早前体。由于它们的大小（SSU过程约为6mda），低温电子断层扫描非常适合于末端旋钮的分析。进一步对已定义状态进行特定的标记，可以精确地定位不同的状态，方便我们的分析。最终，这项研究将导致对SSU和LSU过程体的结构和功能的更详细的了解，并为体内早期核糖体生物发生的迷人机制提供前所未有的见解。