Institute for Biochemistry and Molecular Biology, University of Hamburg, 20146 Hamburg, Germany.
Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
Proc Natl Acad Sci U S A. 2021 Apr 6;118(14). doi: 10.1073/pnas.2022756118.
The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.
Gcn 途径在所有真核生物中都保守，包括人类等哺乳动物，它是整合应激反应 (ISR) 的关键组成部分。Gcn1 作为激酶 Gcn2 的必需效应蛋白，Gcn2 反过来又被核糖体停滞激活，导致 eIF2 磷酸化，随后翻译全局受到抑制。这种适应性反应的微调是由 Rbg2/Gir2 复合物完成的，它是 Gcn2 的负调节因子。尽管有大量可用的生化数据，但关于核糖体上 Gcn 蛋白结构的信息仍然难以捉摸。在这里，我们展示了酵母 Gcn1 蛋白与停滞和碰撞 80S 核糖体复合物的低温电子显微镜结构。Gcn1 与双联体中的两个 80S 核糖体相互作用，使得 Gcn1 HEAT 重复序列跨越碰撞核糖体的 P stalk 区域到前导核糖体的 P stalk 和 A 位区域。前导核糖体处于非旋转状态，A 位有肽酰-tRNA，P 位有空 tRNA，E 位有 eIF5A，A 位因子结合区有 Rbg2/Gir2。相比之下，碰撞核糖体采用旋转状态，A/P 位有肽酰-tRNA，P/E 位有空 tRNA，Mbf1 结合在 40S 亚基上的 mRNA 进入通道旁边。总之，我们的发现揭示了 Gcn2 激活蛋白 Gcn1 与碰撞核糖体的相互作用模式，并深入了解 Gcn2 激活的调节机制。Gcn1 与双联体的结合不仅对 Gcn2 激活的 ISR 具有重要意义，而且对一般的核糖体相关质量控制途径也具有重要意义。
