Laboratoire de Biologie Structurale de la Cellule, BIOC, Ecole polytechnique, CNRS, Institut Polytechnique de Paris, 91128 Palaiseau cedex, France.
Laboratoire de Physique de la Matière Condensée, PMC, Ecole polytechnique, CNRS, Institut Polytechnique de Paris, 91128 Palaiseau cedex, France.
Nucleic Acids Res. 2022 Jun 24;50(11):6532-6548. doi: 10.1093/nar/gkac490.
In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.
在真核生物和古菌中，翻译起始的后期步骤涉及两种起始因子 e/aIF5B 和 e/aIF1A。在真核生物中，eIF5B 在核糖体亚基结合中的作用已经确立，并且获得了显示 eIF5B 结合完整核糖体的结构数据。为了发挥其功能，eIF5B 与 eIF1A 协作。然而，长期以来，人们一直期待获得说明这两个因素如何在小核糖体亚基上相互作用的结构数据。古菌对应物 aIF5B 和 aIF1A 的作用仍有待广泛研究。在这里，我们研究了 Pyrococcus abyssi 翻译起始的后期步骤。使用体外重建的起始复合物和光散射，我们表明结合 GTP 的 aIF5B 可加速亚基结合，而无需 GTP 水解。我们报告了结合 GDP 和 GTP 的 aIF5B 的晶体结构，并分析了与这两种核苷酸状态相关的结构域运动。最后，我们展示了包含 30S 与 mRNA、Met-tRNAiMet、aIF5B 和 aIF1A 结合的起始复合物的 cryo-EM 结构，分辨率为 2.7 Å。结构数据显示了古菌 5B 和 1A 因子如何协同作用，诱导起始 tRNA 形成有利于亚基结合的构象。讨论了翻译起始后期步骤的古菌和真核生物特征。
