Translational GTPases and the energy landscape of the 70S ribosome

翻译GTPases （trGTPases）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。trgtpase通常与GTP构象中的特定核糖体结合，并在其GDP构象中解离。因此，在各种中间体的功能循环中存在复杂的动态分子相互作用和相互依赖。GTP水解和磷酸盐释放可能是确定分子与核糖体相互作用的关键步骤。从亚稳能的角度来看，核糖体可以看作是一台布朗机器，能够在环境温度下对几种构象状态进行采样。trgtpase可以通过构象捕获机制调节能量格局，导致核糖体或因子的明显大规模构象变化。在这里，我们想用多粒子冷冻电镜从结构上分析经典翻译GTPase因子与细菌70S核糖体之间复杂的相互作用。在第二个资助期，tRNA选择和易位以及相应的trGTPase延伸因子EF-Tu和EF-G的工作将继续进行。此外，我们还将分别包括参与翻译起始和终止的翻译GTPases IF2和RF3，以促进对所有四种普遍保守的trGTPases的全面概述。我们将利用冷冻电镜的直接电子检测相机的出现，现在可以在近原子分辨率下获得核糖体复合体的冷冻电镜图。因此，使用低温电镜（P2, P3, P4）的项目之间的持续技术合作将是至关重要的。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trGTPase停滞在核糖体上的70S核糖体的各种复合物的结构和构象模式。含有IF2和其他起始因子的起始复合物的实验将与P6密切合作进行。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。将典型GTPase因子与非典型GTPase因子（P2）的研究结果进行比较，将对翻译型GTPase因子的进化保守性和分化性特征提供重要的见解。此外，对翻译暂停核糖体结合P7的新生链的分析将得到结构研究的支持。

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.