Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.

CNRS UPR9002 Institut de Biologie Moléculaire et Cellulaire Université de Strasbourg Strasbourg 67000 France

INSERM U1212 Institut de Biologie Moléculaire et Cellulaire Université de Strasbourg Strasbourg 67000 France

INSERM U1212 Institut Européen de Chimie et Biologie Université de Bordeaux Pessac 33607 France

Laboratory of Regulation of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Laboratory of Regulation of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic; Charles University Faculty of Science Albertov 6 128 00 Prague 2 Czech Republic

Université de Strasbourg CNRS Plateforme Protéomique Strasbourg Esplanade FRC 1589 Strasbourg 67000 France

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Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation

Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively