eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

Institute of Genetics and Molecular and Cellular Biology CNRS UMR7104 INSERM UMR964 Illkirch France

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

Laboratory of Structural Biology and Cell Signaling Institute of Microbiology of the Czech Academy of Sciences Prague Videnska 1083 142 20 The Czech Republic

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Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3