Structure of the mammalian ribosome as it decodes the selenocysteine UGA codon

Abstract

The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNA^Sec) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l-serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.

Fig. 1.. The mammalian Sec UGA recoding assembly.

(A) Reconstitution of complexes used in this study. (B) Three dimensional (3D) reconstruction of the assembly shown in two views related by ~90° clockwise rotation around the vertical axis. (C) The same views of the final model shown as a surface diagram. (D) The recoding complex as seen from the vantage points of the 40S (left) and 60S (right) subunits. (E) 3D map covering the eEFSec•GTP•Ser-tRNA^Sec•SECIS•SBP2 complex (cartoon). 60S is gray, 40S is sand, eEFSec•GTP is dark red, Ser-tRNA^Sec is dark green, SECIS is blue, SBP2 is dark orange, and the mRNA harboring CrPV IRES is purple.

Fig. 2.. Human eEFSec•GTP•Ser-tRNA^Sec on the 80S ribosome.

(A) Side view of the eEFSec complex (cartoon) as bound to the 80S. (B) D1 and D2 of eEFSec rest against H95 of the 60S and h5 and h14 of the 40S. The Ser-CCA is bound to the Sec-binding pocket. The view is rotated 90° counterclockwise around the vertical axis relative to (A). (C) Close-up view of the Sec-binding pocket with Ser, Ala⁷⁶, and residues of the Sec-binding site shown as stick-and-ball. Dashed lines indicate H-bonds. (D) D4 of eEFSec is between the AAR motif of SECIS and h33 of the 40S. The view is rotated 90° clockwise around the vertical axis relative to (A). (E) Close-up view of the interface between AAR motif, h33, and D4. (F) The Sec UGA readthrough assay based on the luciferase reporter. (G) Ser-tRNA^Sec, but not Ser-tRNA^Ser, supports the SBP2-SECIS–dependent Sec UGA readthrough. Activity levels are presented as fold change over the control sample. White and gray bars correspond to WT and AUCC mutant SECIS, respectively. (H) T242L, T242V, and F522G/Q524G mutants lost the readthrough activity.

Fig. 3.. Human SBP2•SECIS captured on the translating ribosome.

(A) Secondary structure diagram (left) and two views of the 3D structure of the Form II Gpx4 SECIS (right). Major interactions are marked; base pairs are green; and loops are pink, yellow, and light blue. Solid red lines indicate Watson-Crick base pairs, dashed lines are non–Watson-Crick pairs, and red dots are wobble pairs. Color coding is the same as in Fig. 1. (B) RBD binds to the SECIS core (asterisk). The SID interacts with the zinc finger of eS31 (arrow). Contacts between helix II of the SECIS and uS19 and eS31 stabilize the complex. (C) Close-up view of interactions between Arg⁷³¹ of RBD and U1112 of SECIS. (D) N-terminal segment of SID (residues 442–446) forms a parallel β-strand to the C terminus of eS31. (E) Only deletion of residues 403–476 from SBP2 (Δ403–476) abolishes Sec UGA readthrough activity.

Fig. 4.. Eukaryotic selenoprotein elongation at Sec UGA codons.

(A) Cryo-EM map (top) and surface diagram (bottom) of 80S•SECIS•SBP2. (B) Proposed mechanism of Sec UGA recoding in eukaryotes. (1) 80S stalls at an in-frame UGA codon. (2) The RBD binds to the apical loop of SECIS, the mRNA folds over, SBP2•SECIS binds to the 40S, and the N terminus of the SID contacts eS31. This step could occur before ribosome stalling. (3) eEFSec•GTP delivers Sec-tRNA^Sec to the 80S and adopts a preaccommodated state conformation. (4) After GTP hydrolysis, eEFSec dissociates from the assembly, Sec-tRNA^Sec accommodates, and peptide bond synthesis and selenoprotein elongation occur. Steps 2 and 3 are visualized in this work.