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.

在这个项目中，我们想使用冷冻电子层析成像（Cryo-ET）和矫正光和电子显微镜（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。它将在信使RNA（mRNA）中转录的遗传信息转化为细胞的蛋白质组。核糖体的重要性及其结构的复杂性，必不可少的协调和调节的组装。该过程的故障对细胞大多是致命的，可能会引起严重的病理。虽然成熟的核糖体进行了很好的研究，但对其生物发生的结构理解仍然是弹性的。真核生物核糖体生物发生在核橄榄中始于核糖体RNA的前体。与这种RNA前体形成核糖体颗粒相关的核糖体组装因子很多。最近，我们在所谓的米勒树的背景下可视化了这些前斑点颗粒（Neyer等人，自然，2016年）。米勒树是具有核糖体DNA（rDNA）支架的超分子结构，其中包含积极转录RNA聚合酶I（pol I）酶的酶，从中，新生的核糖体RNA（rRNA）链是出现的，并折叠以形成前核糖体颗粒。这些前的丝体颗粒位于米勒树的分支末端，称为末端旋钮。这，米勒树的末端旋钮以准序列的方式可视化前丝体的逐步生成。通过此应用，我们想通过逐步添加蛋白质复合物，在新兴rRNA的5'末端分析末端旋钮。这种分析方法仅是由于在生长上共同组装复合物的定义定位而可行的。米勒树的rn纳斯。在酵母中，末端旋钮的形成是一个两个阶段的过程：早期形成的旋钮将从新生的rRNA链中裂解以形成小核糖体亚基，因此称为小亚基（SSU）过程。后来形成的旋钮将发展为大型核糖体亚基，因此称为大型亚基（LSU）过程。那是旋钮生成的两个阶段过程，代表了最终核糖体亚基的最早前体。由于其大小（SSU过程大约为6个MDA），因此，低温电子断层扫描非常适合分析末端旋钮。进一步关于定义状态的特定标记可以准确地定位不同的状态并促进我们的分析。最终，这项研究应更详细地了解SSU和LSU过程的结构和功能，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。