Distinct pre-initiation steps in human mitochondrial translation

Anas Khawaja,Alexey Amunts,Yuzuru Itoh,Cristina Remes,Olessya Yukhnovets,Henrik Spåhr,Henning Höfig,Joanna Rorbach

doi:10.1038/s41467-020-16503-2

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.

Highlights

Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mitochondrial IF3 (mtIF3), which have diverged from their bacterial counterparts
The canonical bacterial translation begins with the initiation factors IF1, IF2, IF3, and initiator tRNA that assemble on the small ribosomal subunit (SSU) to recognize the mRNA start codon, followed by the joining of the large subunit (LSU)
The preinitiation complex can be formed by adding IF2 to the SSU-IF3 complex in the presence of a nonhydrolyzable GTP analogue (GDPNP), formylmethionyl-tRNAMeti, and mRNA5

Summary

Introduction

Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. 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. To investigate how translation initiation starts in human mitochondria, we analyze here the early involved complexes of the small mitoribosomal subunit using a combination of cryo-EM, fluorescence cross-correlation spectroscopy, and single-molecule fluorescence techniques. Our data reveal two defined steps that lead to the mitochondrial translation initiation, termed mitochondrial preinitiation steps 1 and 2 (mtPIC-1, mtPIC-2), which explains how mitochondria-specific extensions of initiation factors interact with mitoribosomal proteins to govern the mechanism of translation

Methods

Results

Conclusion