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.
- MeSH
- elektronová kryomikroskopie MeSH
- eukaryotický iniciační faktor 1 chemie genetika metabolismus MeSH
- eukaryotický iniciační faktor 3 chemie genetika metabolismus MeSH
- eukaryotický iniciační faktor 5 chemie genetika metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- malé podjednotky ribozomu eukaryotické genetika metabolismus MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus ultrastruktura MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
