The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.hepatitis C virus internal ribosome entry site ͉ subunit organization model3 ͉ top-down analysis of macromolecular complexes ͉ translation regulation ͉ in-solution disruption S ince its identification in the 1970s (1-3), the translation initiation factor eIF3 has been subjected to intense scrutiny. Despite considerable interest, knowledge of many aspects of its structure and function remain elusive because of its overall structural complexity and the lack of facile genetic approaches. It is established, however, that eIF3 is involved in both ribosome biogenesis and protein synthesis in eukaryotes (4). Concerted binding of initiation factors is required to initiate protein synthesis and recruit transfer and messenger RNAs to the 40S subunit before assembly of active ribosomes (5). eIF3 binding may take place initially during this process, together with eIF1 and eIF1A to the 40S, followed by binding of the Met-tRNA i -eIF2-GTP complex. Then mRNA binding, scanning, and AUG recognition occur, enabling the 60S subunit to join to form elongation-competent 80S ribosomes (6). An alternative pathway of initiating protein synthesis, often used by viruses, involves a structured sequence in the 5Ј untranslated region of mRNA known as the internal ribosome entry site (IRES). These sequences promote translation initiation without requiring the full complement of eukaryotic initiation factors (5-7). The hepatitis C virus (HCV) IRES is recognized specifically by the small ribosomal subunit and eIF3 before viral translation initiation, forming stable complexes ...

A key step in translational initiation is the recruitment of the 43S preinitiation complex by the cap-binding complex [eukaryotic initiation factor 4F (eIF4F)] at the 5′ end of messenger RNA (mRNA) to form the 48S initiation complex (i.e., the 48S). The 48S then scans along the mRNA to locate a start codon. To understand the mechanisms involved, we used cryo–electron microscopy to determine the structure of a reconstituted human 48S. The structure reveals insights into early events of translation initiation complex assembly, as well as how eIF4F interacts with subunits of eIF3 near the mRNA exit channel in the 43S. The location of eIF4F is consistent with a slotting model of mRNA recruitment and suggests that downstream mRNA is unwound at least in part by being “pulled” through the 40S subunit during scanning.

Protein synthesis in mammalian cells requires initiation factor eIF3, a ∼750-kilodalton complex that controls assembly of 40 S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5′-cap or an internal ribosome entry site (IRES). Cryo–electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5′-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40 S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40 S , promoting initiation complex assembly.