Abstract
Mitoribosomes have been specialized for protein synthesis in mitochondria. While it is established that mitoribosomes differ between species, a reference model from the Fungi kingdom is missing, and the structural basis for the mitoribosomal function is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate, determined from cryo-EM maps at overall resolution of 2.98-3.09 A. Most of the rRNA expansion segments were modeled, and five additional associated proteins were identified. Mitochondria-specific mS27 is found to have a function of linking two expansion segments. Collectively, the expanded rRNA and mitochondria-specific protein elements coordinate binding of nicotinamide adenine dinucleotide (NAD) in the central protuberance of the large subunit and the ATPase inhibitory factor 1 (IF1) in the small subunit, revealing unprecedented occurrences in translation apparatus. The models of the active fungal mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA translocated with the help of newly identified protein mL108 bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through the exit tunnel involving a series of conformational rearrangements. Finally, an assembly intermediate is found and modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.








