Significance Kinetoplastids is a group of flagellated unicellular eukaryotic parasites including human pathogens, such as Trypanosoma cruzi and Leishmania spp., etiologic agents of Chagas disease and leishmaniasis. They are potentially lethal, affecting more than 20 million people worldwide. Therapeutic strategies are extremely limited and highly toxic. The finding of new molecular targets represents one venue for the development of new therapeutic strategies. In this work, we present the structure of the full mitoribosomes from two kinetoplastids, Leishmania tarentolae and T. cruzi , thus far uncharacterized. We also reveal the structure of an large subunit assembly intermediate harboring 16 different factors. In addition to defining a subset of novel kinetoplastid-specific factors, our results shed light on the mitochondrial ribosomal RNA maturation process.

The 43S preinitiation complex (PIC) assembly requires establishment of numerous interactions among eukaryotic initiation factors (eIFs), Met-tRNAiMet and the small ribosomal subunit (40S). Owing to several differences in the structure and composition of kinetoplastidian 40S compared to their mammalian counterparts, translation initiation in trypanosomatids is suspected to display substantial variability. Here, we determined the structure of the 43S PIC from Trypanosoma cruzi, the Chagas disease parasite, showing numerous specific features, such as different eIF3 structure and interactions with the large rRNA expansion segments 9 S , 7 S and 6 S , and the association of a kinetoplastid-specific ~245 kDa DDX60-like helicase. We also revealed a previously undetermined binding site of the eIF5 C-terminal domain, and terminal tails of eIF2β, eIF1, eIF1A and eIF3 c and d subunits, uncovering molecular details of their critical activities.