Paternoga Helge, Xia Lu, Dimitrova-Paternoga Lyudmila, Li Sihan, Yan Liewei L, Oestereich Malte, Kasvandik Sergo, Nanjaraj Urs Ankanahalli N, Beckert Bertrand, Tenson Tanel, Zaher Hani, Inada Toshifumi, Wilson Daniel N
Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany.
Division of Ribonucleic Acid (RNA) and Gene Regulation, Institute of Medical Science, The University of Tokyo, Minato-Ku, Tokyo 108-8639, Japan.
Proc Natl Acad Sci U S A. 2025 Apr 15;122(15):e2415807122. doi: 10.1073/pnas.2415807122. Epub 2025 Apr 8.
The integrated stress response (ISR) is a central signaling network that enables eukaryotic cells to respond to a variety of different environmental stresses. Such stresses cause ribosome collisions that lead to activation of the kinase Gcn2, resulting in the phosphorylation and inactivation of eukaryotic initiation factor 2 and thereby promoting selective translation of mRNAs to restore homeostasis. Despite the importance of the ISR and intensive study over the past decades, structural insight into how Gcn2 interacts with ribosomal particles has been lacking. Using ex vivo affinity purification approaches, we have obtained a cryoelectron microscopy structure of a yeast Gcn2 dimer in complex with the ribosomal 60S subunit. The Gcn2 dimer is formed by dimerization of the histidine tRNA synthetase-like domains, which establish extensive interactions with the stalk-base and sarcin-ricin loop of the 60S subunit. The C-terminal domain of Gcn2 is also dimerized and occupies the A- and P-site tRNA binding sites at the peptidyl-transferase center of the 60S subunit. Complementary functional studies indicate that binding of Gcn2 to the 60S subunit does not require the coactivators Gcn1 or Gcn20, nor does it lead to phosphorylation of eIF2α. Instead, upon stress, we observe a shift of Gcn2 from the 60S subunit into the colliding ribosome fraction, suggesting that the Gcn2-60S complex represents an inactive stand-by state to enable a rapid redistribution to collided ribosomes, and thereby facilitating a quick and efficient response to stress.
整合应激反应（ISR）是一个核心信号网络，使真核细胞能够对多种不同的环境应激作出反应。此类应激会导致核糖体碰撞，进而激活激酶Gcn2，导致真核起始因子2磷酸化并失活，从而促进mRNA的选择性翻译以恢复体内平衡。尽管ISR很重要且在过去几十年中得到了深入研究，但一直缺乏对Gcn2如何与核糖体颗粒相互作用的结构认识。利用体外亲和纯化方法，我们获得了与核糖体60S亚基结合的酵母Gcn2二聚体的冷冻电镜结构。Gcn2二聚体由组氨酸tRNA合成酶样结构域二聚化形成，该结构域与60S亚基的柄基部和帚曲霉素环建立了广泛的相互作用。Gcn2的C末端结构域也发生二聚化，并占据60S亚基肽基转移酶中心的A位和P位tRNA结合位点。互补的功能研究表明，Gcn2与60S亚基的结合不需要共激活因子Gcn1或Gcn20，也不会导致eIF2α磷酸化。相反，在应激时，我们观察到Gcn2从60S亚基转移到碰撞核糖体组分中，这表明Gcn2-60S复合物代表一种无活性的备用状态，能够快速重新分布到碰撞的核糖体上，从而促进对压力的快速有效反应。
