Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP.
Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP.
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DOI：
10.1038/s41467-021-27415-0
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发表时间：
2021-12-13
影响因子：
16.6
通讯作者：
Korostelev AA
中科院分区：
文献类型：
--
作者：
Carbone CE;Loveland AB;Gamper HB Jr;Hou YM;Demo G;Korostelev AA
在翻译过程中，一个保守的GTPase延伸因子-细菌中的ef - g或真核生物中的eEF2 -通过核糖体转运tRNA和mRNA。EF-G被认为是一种推动tRNA和mRNA运动的柔性马达，作为一种刚性的爪子，偏向核糖体重排导致的单向易位，或者通过马达和爪子样机制的各种组合。使用时间分辨低温电镜，我们在没有抑制剂的情况下可视化了gtp催化的易位，以接近原子的分辨率捕获了核糖体•EF-G中间体的难以捉摸的结构。在易位之前，EF-G结合在肽基trna附近，而旋转的30S亚基稳定了EF-G GTPase中心。反向30S旋转释放Pi，使肽基trna和EF-G易位~20 Å。另外一个4-Å易位启动了瞬态核糖体状态下的EF-G解离，具有高度旋转的30S头。这些结构显示了几乎刚性的EF-G如何将固有的和自发的核糖体动力学纠正为tRNA-mRNA易位，而GTP水解和Pi释放驱动EF-G解离。EF-G沿mRNA驱动核糖体易位。时间分辨低温电镜捕获的易位与EF-G•GTP -无抑制剂-揭示了EF-G如何利用核糖体波动驱动易位和GTP水解在正确的时刻离开。
During translation, a conserved GTPase elongation factor—EF-G in bacteria or eEF2 in eukaryotes—translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation. EF-G drives ribosomal translocation along mRNA. Time-resolved cryo-EM captured translocation with EF-G•GTP—without inhibitors—revealing how EF-G uses ribosome fluctuations to drive translocation and GTP hydrolysis to leave at the right moment.
影响因子：
2.8
作者：
Fenwick MK;Ealick SE
通讯作者：
Ealick SE