Structure of Gcn1 bound to stalled and colliding 80S ribosomes

Abstract

Protein synthesis is essential to cells and requires a constant supply of nutrients. Amino acid starvation leads to accumulation of uncharged tRNAs that promote ribosomal stalling, which is sensed by the protein kinase Gcn2, together with its effector proteins, Gcn1 and Gcn20. Activation of Gcn2 phosphorylates eIF2, leading to a global repression of translation. Fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, which is a negative regulator of Gcn2. Despite the wealth of biochemical data, structures of Gcn proteins on the ribosome have 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 A-site region of the lead ribosome. The lead ribosome is stalled in a non-rotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, as well as Rbg2/Gir2 located 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 provide a structural basis for Rbg2/Gir2 repression of Gcn2, and also reveal that colliding disomes are the substrate for Gcn1 binding, which has important implications not only for Gcn2-activated stress responses, but also for general ribosome quality control (RQC) pathways.

Conserved heterodimeric GTPase Rbg1/Tma46 promotes efficient translation in eukaryotic cells

“…In contrast, amino acid sequences of Dfrp1 and Dfrp2, the Drg family regulatory proteins, are quite different except for their DFRP domain ( Supplemental Figure 1 ). This suggests that the two Dfrp proteins are involved in different protein-protein interactions to engage Drg proteins to the ribosome, as also seen in yeast colliding ribosomes involve Gcn1 43 . Our structure explains earlier experimental observations that Dfrp1 specifically binds Drg1, and Dfrp2 binds to Drg2 preferentially 12 .…”

Frameshifting at collided ribosomes is modulated by elongation factor eEF3 and by Integrated Stress Response regulators Gcn1 and Gcn20

“…Induction of global stress-response pathways is also mediated through ribosome collisions. In both yeast and humans, ribosome collisions result in activation of the integrated stress response (ISR) kinase Gcn2, resulting in a global reduction in translation initiation (Meydan and Guydosh 2020;Wu et al 2020;Pochopien et al 2021;Yan and Zaher 2021). The observation that key effectors of the ISR, Gcn1, Gcn20, Rbg2 and Gir2, bind collided ribosomes with Mbf1 (Pochopien et al 2021) likely explains a previous observation that Mbf1 also modulates the induction of the ISR in yeast (Takemaru et al 1998).…”

“…In both yeast and humans, ribosome collisions result in activation of the integrated stress response (ISR) kinase Gcn2, resulting in a global reduction in translation initiation (Meydan and Guydosh 2020;Wu et al 2020;Pochopien et al 2021;Yan and Zaher 2021). The observation that key effectors of the ISR, Gcn1, Gcn20, Rbg2 and Gir2, bind collided ribosomes with Mbf1 (Pochopien et al 2021) likely explains a previous observation that Mbf1 also modulates the induction of the ISR in yeast (Takemaru et al 1998). In humans, the ribosomeassociated MAPKKK hZAKα autophosphorylates during ribosome collisions, resulting in activation of stress-activated protein kinases p38 and cJun which respectively cause cell cycle arrest and apoptosis (Sinha et al 2020;Wu et al 2020).…”

“…Second, crucial regulators of quality control responses (Asc1/hRACK1, uS3 and Mbf1/hEDF1) occupy central positions in structures of collided ribosomes (disomes and trisomes), supporting their role in regulation of quality control responses. For instance, yeast Asc1 (hRACK1) (Kuroha et al 2010) and uS3 (Simms et al 2018;Wang et al 2018) reside at the 40S-40S interface of collided ribosomes (Matsuo et al 2017;Juszkiewicz et al 2018;Ikeuchi et al 2019;Sinha et al 2020), and Mbf1 (hEDF1) (Wang et al 2018;Simms et al 2019;Juszkiewicz et al 2020a;Sinha et al 2020) interacts with uS3 on the colliding ribosome (Sinha et al 2020;Pochopien et al 2021) Third, crucial regulators of quality control are specifically recruited to collided ribosomes, rather than monosomes. For instance, both Hel2 and hZNF598 act preferentially on disomic or trisomic ribosomes in vitro (Juszkiewicz et al 2018;Ikeuchi et al 2019;Matsuo et al 2020), and both hZNF598 and hEDF1 are specifically enriched on nuclease-resistant ribosome multimers compared to their relative abundance on monosomes (Juszkiewicz et al 2020a;Sinha et al 2020).…”

“…For instance, both Hel2 and hZNF598 act preferentially on disomic or trisomic ribosomes in vitro (Juszkiewicz et al 2018;Ikeuchi et al 2019;Matsuo et al 2020), and both hZNF598 and hEDF1 are specifically enriched on nuclease-resistant ribosome multimers compared to their relative abundance on monosomes (Juszkiewicz et al 2020a;Sinha et al 2020). Gcn1, an essential component of the ISR, also specifically binds collided ribosomes with Mbf1 (Pochopien et al 2021;Yan and Zaher 2021). Fourth, frameshifting at CGA codon repeats in yeast, which occurs when Mbf1 or uS3 proteins are mutated (Wang et al 2018), is critically dependent upon ribosome density on the mRNA and the position of the CGA codon repeats relative to the AUG start, as expected if collisions are required for the frameshift (Wolf and Grayhack 2015;Simms et al 2019).…”