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.

中文翻译：

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与大 60S 核糖体亚基复合体的 Gcn2 二聚体的结构


整合应激反应 （ISR） 是一个中央信号转导网络，使真核细胞能够对各种不同的环境应激做出反应。这种压力会导致核糖体碰撞，导致激酶 Gcn2 激活，导致真核起始因子 2 的磷酸化和失活，从而促进 mRNA 的选择性翻译以恢复体内平衡。尽管 ISR 和过去几十年的深入研究很重要，但一直缺乏关于 Gcn2 如何与核糖体颗粒相互作用的结构见解。使用离体亲和纯化方法，我们获得了与核糖体 60S 亚基复合的酵母 Gcn2 二聚体的冷冻电子显微镜结构。Gcn2 二聚体是由组氨酸 tRNA 合成酶样结构域的二聚化形成的，这与 60S 亚基的茎碱基和沙蛋白-蓖麻毒素环建立了广泛的相互作用。Gcn2 的 C 端结构域也呈二聚化状态，并占据 60S 亚基肽基转移酶中心的 A 位点和 P 位点 tRNA 结合位点。互补功能研究表明，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，在应激下，我们观察到 Gcn2 从 60S 亚基转移到碰撞核糖体部分，这表明 Gcn2-60S 复合物代表一种无活性的待机状态，能够快速重新分布到碰撞的核糖体，从而促进对应激的快速有效反应。