Distinct pre-initiation steps in human mitochondrial translation

Abstract

Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.

Fig. 1. Structure of the human mitochondrial preinitiation translation complex (mtPIC-2).

a Surface representation of the mtSSU with mtIF3 (orange) and mtIF2 (blue). Empty tRNA-binding sites and mRNA channel are indicated. The binding of mtIF2 is accomplished due to mtIF3 NTD restricting the mtSSU head movement. b Relative positions of mtIF3 colored by domains (NTD purple, linker dark gray, CTD orange, CTE red) and mtIF2.

Fig. 2. Multiple interactions of mtIF3 with the mtSSU.

a Domain organization of mtIF3 on mtSSU with mitochondria-specific CTE (red) positioned outward from mtSSU. b Zoom-in panels for each of the mtIF3 domains featuring interactions with mtSSU. c Schematic representation of the mtIF3 with the corresponding color-code. NTE is disordered in the structure.

Fig. 3. Binding of mtIF3 and fMet-tRNA^fMet_i to the mtSSU is mutually exclusive.

a Two-color fluorescence cross-correlation spectroscopy analysis of the initiation complex components. The mtSSU was incubated with an excess of Cy3-fMet-tRNA^fMet_i, Cy5-mtIF3, mtIF2(GTP), and Atto390-mRNA, then purified by a sucrose gradient. Products may contain unlabeled mtIF2. b The functionality of mtPIC was validated by the ability to form a complete initiation complex when incubated with an excess of mtLSU, Cy3-fMet-tRNA^fMet_i, Atto390-mRNA, and unlabeled mtIF2 (GTP). After purification of the monosome, detection of both Cy3 and Atto390 fluorophores confirmed binding of mRNA and tRNA. Source data are provided as a Source Data file.

Fig. 4. Principal component analysis of the mtSSU head motion suggests a function for the mitoribosomal protein mS37 in restricting the swiveling.

a–c The relative orientation of the head to the body is described by 12 eigenvectors, representing translation and rotation in each particle. The eigenvectors 1, 2, and 3 were found to mainly contribute to the head motion, as indicated. d Histogram of the variances explained by the 12 eigenvectors. e, f The 1st to 3rd eigenvalues of randomly chosen particles represented by dots are plotted against each other, showing non-gaussian distribution for the swiveling motion. The particles are classified based on the values of eigenvector 1 (rectangles). g, h Comparison between the reconstructions of the classified particles. The closed conformation (Class 1) exhibits weaker density for the mtIF3-NTD interacting with mS37 and uS7m, compared to the other conformations with higher particle numbers.

Fig. 5. Contacts between mtIF2 and mtSSU in mtPIC-2, and conformational changes upon mtLSU association.

a Comparison of mtIF2 binding in mtPIC-2 with the complete initiation complex (PDB ID: 6GAW). Upon association of the mtLSU (blue), required for complete initiation complex, the mtIF2-domain IV moves towards the mtSSU. The conformational change is represented by the difference vectors for each C_α atom of domain IV. For the other domains, the flexibility is represented through ribbon thickness according to B-factor. b Zoom-in panels showing contacts of the mtIF2-specific insert and linker domain with the mtSSU. c Schematic representation of the mtIF2 with the corresponding color-code to the conformational changes.

Fig. 6. Binding of leaderless mt-mRNA to the monosome.

a An optical tweezers setup to study mitochondrial translation initiation. RNA construct containing the first 51 nucleotides of MT-CO2 mRNA was ligated to biotinylated lambda DNA, allowing the 5′-end accessible for mitoribosome binding. b In order to monitor both mitoribosomal subunits, mtLSU and mtSSU were labeled with Cy5 and Atto488, respectively, and initiation reactions were made by incubating either double-labeled monosome (b, 1.) or Cy5-labeled mtSSU (b, 3.) with the DNA/RNA hybrid, mtIF3, mtIF2 (GTP), and fMet-tRNA^Met_i. An RNA construct without start codon was used as a negative control (b, 2.). c Representative images showing binding of the mitoribosomal subunits tested by simultaneous dual-color confocal imaging. d Quantification of the binding events. The number of detected fluorescently labeled species were normalized to the number of trapped imaged DNA. Bars represent mean ± SD. Source data are provided as a Source Data file.

Fig. 7. Model of mitochondrial translation initiation and comparison with the bacterial system.

a Translation initiation in mammalian mitochondria. The various structures are summarized in the order that represents a possible initiation pathway in which mtIF2 binding precedes mtLSU and tRNA binding. In this pathway, mtIF3 and mS37 stabilize the mtSSU head for the accommodation of mtIF2. Joining of the mtLSU may result in the conformational change of mtIF2 and GTPase activation that leads to tRNA and mRNA accommodation. Alternatively, transient binding of both mRNA and tRNA (not detected with our techniques) to the mtSSU after mtIF3 departure precede recruitment of the mtLSU. b Translation initiation in bacteria. Binding of canonical mRNA, fMet-tRNA^Met_i and initiator factors precede subunit joining and formation of the complete initiation complex.