Translational GTPases and the energy landscape of the 70S ribosome

翻译GTP酶(TrGTP Ase)是一组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控制核糖体。TrGTP酶通常在其GTP构象中结合到核糖体的特定状态，并在其GDP构象中解离。因此，在各种中间体的功能周期中，存在复杂的动态分子相互作用和相互依赖。GTP的水解和磷酸盐的释放可能是确定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，核糖体可以看作一个布朗机器，能够在常温下采样几种构象状态。trGTP酶可以通过构象捕获机制调节能量景观，导致核糖体或因子发生明显的大范围构象变化。在这里，我们想用多粒子低温EM技术，从结构上分析规范的翻译GTP酶因子与细菌70S核糖体之间的复杂相互作用。在第二个资助期，将继续tRNA选择和易位以及各自的trGTP酶延长因子EF-Tu和EF-G的工作。此外，我们还将包括分别参与翻译起始和终止的翻译GTP酶IF2和Rf3，以便于全面概述所有四种普遍保守的trGTP酶。我们将利用低温电子显微镜的直接电子探测相机的出现，这使得以近原子分辨率获得核糖体复合体的低温电子显微镜图谱成为可能。因此，使用低温EM(P2、P3、P4)的项目之间的持续技术协作将至关重要。这将使我们能够在各种实验条件下，例如使用抗生素或非水解性GTP类似物，解决70年代核糖体与trGTP酶停滞在核糖体上的各种络合物的结构和构象模式。含有IF2和其他引发因子的引发络合物的实验将与P6密切合作进行。需要进行功能研究并得到P5、P6和P7的进一步支持，才能在功能范围内解释结构工作。将典型GTP酶因子的研究结果与非典型GTP酶因子(P2)的研究结果进行比较，将对揭示翻译GTP酶因子的进化保守性和发散性有重要意义。此外，P7对翻译暂停的核糖体结合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.