Choreography of human mitochondrial leaderless mRNA translation initiation

It remains unclear how human mitochondrial ribosomal subunits assemble into an elongation-competent 55S particle on mRNAs devoid of 5’ leader sequences. Here, we reconstituted and directly tracked human mitochondrial translation initiation using real-time single-molecule fluorescence spectroscopy. Corroborated with cryo-EM structural analysis, we show that the initiation factor mtIF2 and initiator fMet-tRNA ^Met are loaded to the 28S subunit to drive mRNAs binding via 5’ start codon recognition. This enables sequential loading of the two ribosomal subunits onto the leaderless mRNA to initiate. In parallel, a preassembled 55S monosome can also be loaded with mtIF2 and fMet-tRNA ^Met to initiate on the mRNA. Both initiation pathways yield active complexes to enter translation elongation, which is gated by mtIF2. The monosome loading pathway can initiate promiscuously with non-formylated Met-tRNA ^Met , thus its usage may under tight regulation in cells, e.g. by mtIF3. Our work provides a dynamic framework for the distinct human mitochondrial translation initiation.