Abstract
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.
Highlights
All living cells must adapt to a variety of different environmental stresses in a rapid and efficient way to survive
We were not able to visualize Gcn1 on the ribosomes from samples treated with 3-AT, whereas a low- resolution cryo-electron microscopy (EM) reconstruction of the untreated Gcn20-TAP sample revealed that a minor (5%) subpopulation of ribosomes contained an additional tube-like density, which we assigned to Gcn1 (SI Appendix, Fig. S2)
Under conditions of amino acid starvation, the binding of deacylated transfer RNAs (tRNAs) in the A-site of the ribosome causes translational stalling, which in turn increases the frequency of ribosome collisions and disome formation [35, 36] (Fig. 4A)
Summary
All living cells must adapt to a variety of different environmental stresses in a rapid and efficient way to survive. The central region of Gcn is highly homologous to the N-terminal HEAT repeat region of the eukaryotic elongation factor 3 (eEF3) [3] (Fig. 1A), and overexpression of eEF3 represses Gcn activity, suggesting that Gcn and eEF3 have overlapping binding sites on the ribosome [6]. A region (residues 2052 to 2428) within the C terminus of Gcn mediates direct interaction with the N-terminal RWD domain of Gcn (Fig. 1A) [4, 9, 10] Mutations within these regions of either Gcn (F2291L or R2259A) or Gcn (Y74A) disrupt the Gcn1–Gcn interaction, resulting in loss of both eIF2 phosphorylation and derepression of Gcn translation [4, 10]. The structure provides insight into the regulation of Gcn activation in yeast and has implications for the interplay between the RQC and ISR pathways in eukaryotic cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
