Translational GTPases and the energy landscape of the 70S ribosome

翻译 GTP 酶和 70S 核糖体的能量景观

• 批准号: 220066510
• 负责人: Professor Dr. Christian M. T. Spahn
• 金额: --
• 依托单位: Institut für Medizinische Physik und Biophysik (IMPB)
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/220066510?language=en
• 关键词: Translational; GTPases; energy; landscape; 70S

Translational GTPases (trGTPases) constitute an important group of external translation factors. They control and steer the ribosome during all four phases of protein synthesis. trGTPases usually bind to a specific state of the ribosome in their GTP conformation and dissociate in their GDP conformation. Consequently, there are complex dynamic molecular interactions and interdependences during the functional cycle within the various intermediates. GTP hydrolysis and phosphate release may be critical steps in defining the molecular interplay with the ribosome. In the metastable energy landscape view the ribosome can be regarded as a Brownian machine capable of sampling several conformational states at ambient temperature.trGTPases can tune the energy landscape resulting in apparent large-scale conformational changes in the ribosome or the factor by a conformational capture mechanism. Here we want to structurally analyze the complex interplay between canonical translational GTPase factors with the bacterial 70S ribosome using multiparticle cryo-EM. In the second funding period the work on tRNA selection and translocation and the respective trGTPase elongation factors EF-Tu and EF-G shall be continued. Furthermore, we will include also the translational GTPases IF2 and RF3 involved in translation initiation and termination, respectively to facilitate a comprehensive overview of all four universally conserved trGTPases. We will capitalize on the advent of direct electron detection cameras for cryo-EM that now makes it feasible to obtain cryo-EM maps of ribosomal complexes at near-atomic resolution. Hereby the continued technical collaboration among the project using cryo-EM (P2, P3, P4) will be of crucial importance. This will allow us to solve the structure and conformational modes of various complexes of the 70S ribosomes with a trGTPase stalled on the ribosome in a variety of experimental conditions, e.g. using antibiotics or non-hydrolysable GTP analogues. Experiments on initiation complexes containing IF2 and other initiation factors will be done in close collaboration with P6. Functional studies and further support from P5, P6 and P7 will be required for an interpretation of the structural work in a functional context. The comparison of results for canonical GTPase factors with studies on non-canonical GTPase factors (P2) will yield important insights into the evolutionary conserved and divergent features of translational GTPase factors. Furthermore, the analysis on translationally paused ribosome-bound nascent chains by P7 will be supported by structural studies.

翻译GTP酶（translational GTP ases，trGTP ases）是一类重要的外部翻译因子。它们在蛋白质合成的所有四个阶段控制和引导核糖体。trGTP酶通常以其GTP构象结合到核糖体的特定状态，并以其GDP构象解离。因此，在各种中间体的功能循环过程中存在复杂的动态分子相互作用和相互依赖性。GTP水解和磷酸盐释放可能是定义与核糖体的分子相互作用的关键步骤。在亚稳态能量景观观点中，核糖体可以被视为能够在环境温度下采样几种构象状态的布朗机器。trGTP酶可以通过构象捕获机制调节能量景观，导致核糖体或因子中明显的大规模构象变化。在这里，我们想用多粒子冷冻电镜从结构上分析典型的翻译GTdR因子与细菌70 S核糖体之间的复杂相互作用。在第二个资助期内，将继续开展tRNA选择和易位以及相应的trGTactin延伸因子EF-Tu和EF-G的工作。此外，我们还将包括参与翻译起始和终止的翻译GTP酶IF 2和RF 3，以便于全面概述所有四种普遍保守的trGTP酶。我们将利用直接电子检测相机的出现，冷冻EM，现在可以获得近原子分辨率的核糖体复合物的冷冻EM地图。因此，使用冷冻EM（P2，P3，P4）的项目之间的持续技术合作至关重要。这将使我们能够在各种实验条件下，例如使用抗生素或不可水解的GTP类似物，解决70 S核糖体与在核糖体上停滞的trGTP酶的各种复合物的结构和构象模式。将与P6密切合作，对含有IF 2和其他引发因子的引发复合物进行实验。功能研究和P5，P6和P7的进一步支持将需要在功能背景下解释结构工作。经典的GTdR因子与非经典的GTdR因子（P2）的研究结果的比较将产生重要的见解的翻译GTdR因子的进化保守和分歧的功能。此外，通过P7对断裂暂停的核糖体结合新生链的分析将得到结构研究的支持。

项目成果

Ribosomal Chamber Music: Toward an Understanding of IRES Mechanisms.

• DOI: 10.1016/j.tibs.2017.06.002
• 发表时间: 2017-08
• 期刊: Trends in biochemical sciences
• 影响因子: 13.8
• 作者: Hiroshi Yamamoto;A. Unbehaun;C. Spahn

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

核糖体结合丙型肝炎病毒内部核糖体进入位点 RNA 的分子结构

• DOI: 10.15252/embj.201592469
• 发表时间: 2015
• 期刊: The EMBO Journal
• 作者: Yamamoto;Collier;Loerke;Schmidt;Sprink;Yamamoto;Mielke;Bürger;Shaikh;Dabrowski;Hildebrand;Scheerer;C.M.T.
• 通讯作者: C.M.T.

Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA

HCV-IRES RNA 上具有起始因子 5B 的哺乳动物 80S 起始复合物的结构

• DOI: 10.1038/nsmb.2859
• 发表时间: 2014
• 期刊: Nature Structural &Molecular Biology
• 作者: Yamamoto;Unbehaun;Loerke;Behrmann;Collier;Bürger;Mielke;C.M.T.
• 通讯作者: C.M.T.

Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation

• DOI: 10.1073/pnas.1320387110
• 发表时间: 2013-12-24
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Ramrath, David J. F.;Lancaster, Laura;Spahn, Christian M. T.
• 通讯作者: Spahn, Christian M. T.