Zeng Fuxing, Chen Yanbo, Remis Jonathan, Shekhar Mrinal, Phillips James C, Tajkhorshid Emad, Jin Hong
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208-3500, USA.
Nature. 2017 Jan 26;541(7638):554-557. doi: 10.1038/nature21053. Epub 2017 Jan 11.
Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs that lack stop codons is one of the co-translational quality control pathways. In many bacteria, ArfA recognizes stalled ribosomes and recruits the release factor RF2, which catalyses the termination of protein synthesis. Although an induced-fit mechanism of nonstop mRNA surveillance mediated by ArfA and RF2 has been reported, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report an electron cryo-microscopy structure of ArfA and RF2 in complex with the 70S ribosome bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to enable RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Furthermore, binding of ArfA and RF2 induces conformational changes in the ribosomal decoding centre that are similar to those seen in other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 to adopt a catalytically competent conformation for peptide release. Our findings provide a framework for understanding recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.
当出现有缺陷的翻译事件时，质量控制机制会进行适当干预，以维护蛋白质合成的完整性。对缺乏终止密码子的信使核糖核酸（mRNA）上进行翻译的核糖体进行拯救，是共翻译质量控制途径之一。在许多细菌中，ArfA识别停滞的核糖体并招募释放因子RF2，RF2催化蛋白质合成的终止。尽管已经报道了由ArfA和RF2介导的无义mRNA监测的诱导契合机制，但负责该机制的ArfA与RF2在核糖体中的分子相互作用尚不清楚。在这里，我们报告了ArfA和RF2与结合无义mRNA的70S核糖体形成复合物的冷冻电镜结构。该结构与我们的动力学和生化数据一致，揭示了使ArfA能够特异性地将RF2而非RF1招募到核糖体中，并使RF2在这种共翻译质量控制途径中释放截短的蛋白质产物的分子相互作用。ArfA带正电荷的C末端结构域锚定在核糖体的mRNA进入通道中。此外，ArfA和RF2的结合诱导核糖体解码中心的构象变化，这与其他涉及蛋白质的解码过程中观察到的变化相似。ArfA N末端结构域与RF2中残基之间的特异性相互作用有助于RF2采用催化活性构象以释放肽段。我们的发现为理解新蛋白质对核糖体翻译状态的识别提供了一个框架，并扩展了我们对核糖体解码潜力的认识。
