Structural insights into unique features of the human mitochondrial ribosome recycling

Abstract

Mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation (ATP generation). Despite their common ancestry with bacteria, the composition and structure of the human mitoribosome and its translational factors are significantly different from those of their bacterial counterparts. The mammalian mitoribosome recycling factor (RRF_mt) carries a mito-specific N terminus extension (NTE), which is necessary for the function of RRF_mt Here we present a 3.9-Å resolution cryo-electron microscopic (cryo-EM) structure of the human 55S mitoribosome-RRF_mt complex, which reveals α-helix and loop structures for the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoribosome. These include ribosomal RNA segments that constitute the peptidyl transferase center (PTC) and those that connect PTC with the GTPase-associated center and with mitoribosomal proteins L16 and L27. Our structure reveals the presence of a tRNA in the pe/E position and a rotation of the small mitoribosomal subunit on RRF_mt binding. In addition, we observe an interaction between the pe/E tRNA and a mito-specific protein, mL64. These findings help understand the unique features of mitoribosome recycling.

Keywords: 55S–RRFmt complex; cryo-EM structure; human mitochondrial RRF; mito-specific interactions; mito-specific sequence.

Fig. 1.

Cryo-EM structure of the human 55S mitochondrial ribosome in complex with RRF_mt. (A) 3D cryo-EM map of the 55S mitoribosome-RRF_mt complex as seen from the subunit–subunit interface side, with segmented densities corresponding to the small subunit (28S, yellow), large subunit (39S, blue), and RRF_mt (orange, red, and pink). (B) Molecular interpretation of the cryo-EM map shown in A. A darker shade of yellow differentiates the 28S ribosomal proteins from the 12S rRNA, whereas a lighter shade of blue differentiates the 39S ribosomal proteins from the 16S rRNA. Landmarks of the 28S subunit: h, head; b, body. Landmarks of the 39S subunit: CP, central protuberance. (C and D) Molecular model of RRF_mt as derived from the cryo-EM map, showing well-resolved densities for both the conserved domains I and II (orange) and the NTE (red). The better-resolved segments of RRF_mt with sidechains are shown in D. (E) Domain organization of the human RRF_mt. The structurally resolved and unresolved portions of the NTE are depicted in red and pink colors, respectively (SI Appendix, Fig. S8).

Fig. 2.

Binding positions of bound RRF_mt, mRNA, and A- and P-site tRNAs on the mitoribosome. RRF_mt binding would be in direct steric clash with the acceptor arm of both aminoacyl- and peptidyl- tRNAs on the large subunit. Coordinates of the mRNA (dark brown) and tRNAs (pink and light blue) were derived from the structure of porcine mitoribosome (25) (PDB ID: 5AJ4). The conserved RRF_mt domains and its NTE are colored as in Fig. 1. The NTE of RRF_mt lies in close proximity to the functionally important and conserved A (green) and P loops (dark blue). A thumbnail to left depicts an overall orientation of the 55S mitoribosome, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid positions of ligands. Landmarks on the thumbnail: h, head, and b, body of the 28S subunit, and CP, central protuberance of the 39S subunit.

Fig. 3.

Interactions of structurally conserved domains of RRF_mt with the 55S mitoribosome. (A) Contacts between the domain I (orange) and the P loop, the 16S rRNA H80 (blue). (B) Interactions between the domain I and H71 (olive green) of the 16S rRNA. (C) Contacts between the ribosomal protein S12 (magenta) and RRF_mt domain II. Thumbnails to left depict overall orientations of the 55S mitoribosome, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid positions of RRF_mt. Landmarks on the thumbnails are same as in Fig. 2.

Fig. 4.

Mito-specific Interactions between the NTE of RRF_mt and the 39S mitoribosomal subunit proteins. (A) The N-terminal region of L27 (purple) in the human 55S–RRF_mt complex has shifted by ∼9 Å toward the peptidyl tRNA-binding site compared with the positions of L27 in the porcine (gray) (25) (PDB ID: 5AJ4) and empty human (green) (24) (PDB ID: 3J9M) mitoribosomes, to interact with the NTE (red) of RRF_mt. In this conformation, L27 would block the binding of tRNA in the 39S P site. Mito-specific interactions between the NTE and mitoribosomal proteins are shown in panels (B) L27 and (C) L16 (dark cyan). Thumbnails to the left depict overall orientations of the 55S mitoribosome, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid positions of ligands. Landmarks on the thumbnails are same as in Fig. 2.

Fig. 5.

Mito-specific interactions between the NTE of RRF_mt and the 16S rRNA components of the 39S subunit. (A) The NTE (red) of RRF_mt interacts with several functionally important segments of the peptidyl-transferase center (PTC), including the P loop (blue), H89 (yellow), H90 (pink), and A loop (H92) (green). (B) Mito-specific interactions of the NTE with the nucleotide bases of H89, H90, and the A loop. Color codes are same as in A. Thumbnails on the top depict overall orientations of the 55S mitoribosome in A and B, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid positions of RRF_mt. Landmarks on the thumbnails are same as in Fig. 2. (C) Overall location of the NTE on the 39S subunit (semitransparent blue), as seen from the subunit’s interface side, with the rRNA components of PTC that extend up to the GTPase-associated center (GAC), which includes the α-sarcin-ricin stem loop (H95). Both PTC and GAC are depicted as dashed circles. (D) Secondary structure of the PTC region of the 16S rRNA, highlighting helices that are color-coded as in A–C, and interacting nucleotides with red ovals.