Structure of Gcn1 bound to stalled and colliding 80S ribosomes.
Structure of Gcn1 bound to stalled and colliding 80S ribosomes.
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DOI：
10.1073/pnas.2022756118
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发表时间：
2021-04-06
影响因子：
11.1
通讯作者：
Wilson DN
中科院分区：
文献类型：
--
作者：
Pochopien AA;Beckert B;Kasvandik S;Berninghausen O;Beckmann R;Tenson T;Wilson DN
越来越多的证据表明，核糖体之间的碰撞代表了激活多种应激途径的细胞信号，如核糖体相关质量控制（RQC）、核糖体毒性应激反应和综合应激反应（ISR）。在这里，我们通过展示ISR蛋白Gcn1的天然复合物与前导停滞核糖体和随后碰撞核糖体相互作用的低温电子显微镜结构，说明了单个蛋白质如何监测二体内的两个核糖体。该结构为酵母中Gcn2活化的调控提供了见解，并对真核细胞中RQC和ISR途径之间的相互作用具有启示意义。Gcn通路在所有真核生物中都是保守的，包括人类等哺乳动物，它是综合应激反应（ISR）的关键部分。Gcn1作为Gcn2激酶的重要效应蛋白，Gcn2激酶反过来被停滞的核糖体激活，导致eIF2的磷酸化和随后的翻译的全局抑制。这种自适应反应的微调是由Rbg2/Gir2复合物完成的，Rbg2/Gir2是Gcn2的负调节因子。尽管有丰富的生物化学数据，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蛋白与停滞和碰撞的80S核糖体复合物的低温电镜结构。Gcn1与二体内的两个80S核糖体相互作用，使得Gcn1 HEAT重复序列从碰撞核糖体的P-stalk区域跨越到前导核糖体的P-stalk和A-site区域。导联核糖体处于非旋转状态，肽基tRNA位于a位点，未带电tRNA位于p位点，eIF5A位于e位点，Rbg2/Gir2位于a位点因子结合区。相比之下，碰撞核糖体与a /P-位点的肽基trna、P/ e位点的未带电trna和40S亚基上mRNA进入通道附近的Mbf1结合，采用旋转状态。总的来说，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，并为Gcn2激活的调控提供了见解。Gcn1与二体的结合不仅对gcn2激活的ISR具有重要意义，而且对一般核糖体相关的质量控制途径也具有重要意义。
There is growing evidence that collisions between ribosomes represent a cellular signal for activating multiple stress pathways, such as ribosome-associated quality control (RQC), the ribotoxic stress response, and the integrated stress response (ISR). Here we illustrate how a single protein can monitor both ribosomes within a disome, by presenting a cryo-electron microscopy structure of a native complex of the ISR protein Gcn1 interacting with both the leading stalled ribosome and the following colliding ribosome. The structure provides insight into the regulation of Gcn2 activation in yeast and has implications for the interplay between the RQC and ISR pathways in eukaryotic cells. 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.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
9.2
作者：
Deng, J;Harding, HP;Sonenberg, N
通讯作者：
Sonenberg, N
影响因子：
14.9
作者：
Francis, Sandrea M.;Gas, Maria-Eugenia;Seraphin, Bertrand
通讯作者：
Seraphin, Bertrand
影响因子：
64.8
作者：
Andersen, Christian B. F.;Becker, Thomas;Beckmann, Roland
通讯作者：
Beckmann, Roland
影响因子：
16
作者：
Juszkiewicz S;Chandrasekaran V;Lin Z;Kraatz S;Ramakrishnan V;Hegde RS
通讯作者：
Hegde RS