Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Abstract

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.

eIF3b and eIF3i relocate together to the ribosomal subunit interface during translation initiation and modulate start codon selection

“…A recent study using advanced mass spectrometry revealed that the yeast eIF3 complex in solution adopts a globular 3D structure, with the WD40 domains/β-propellers of eIF3b and eIF3i, the RRMs of eIF3b and eIF3g and the N-and C-terminal ends of eIF3a and eIF3c all exposed to the surface. Interestingly, the PCI domains of eIF3a and eIF3c do not interact with each other in the free eIF3 complex (Zeman et al, 2019). This stands in contrast to the interaction between the two PCI domains observed in the structures of eIF3 present in various conformational states of the PIC, wherein the eIF3 complex seems to have undergone a large conformational change on binding to the 40S subunit (Hashem et al, 2013) (Aylett et al, 2015) 4 (Llácer et al, 2015) (Llácer et al, 2018) (des Georges et al, 2015) (Eliseev et al, 2018).…”

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

“…With yeast eIF3a as the FOI, we observed an ~50 fold increase in the 80S FP coverage along the eIF3b (PRT1) mRNA for eIF3a::80S FPs versus unselected 80S FPs starting near nucleotide 580 and persisting until the end of the CDS ( Figure 7B). The onset of this increase is ~30 codons downstream of the CDS region that encodes the eIF3b RNA recognition motif (RRM; nucleotides 237 -477), which is one of the two main interacting domains for eIF3a within the eIF3 complex ( Figure S1A) (Valášek et al, 2002;Zeman et al, 2019). As the ribosome exit tunnel accommodates ~24 amino acids in extended conformation and ~38 amino acids in α-helical conformation (Bhushan et al, 2010), the nascent RRM should have just emerged from the ribosome when an increase in FP coverage is seen.…”

“…These schematics illustrate similarities and differences between budding yeast and mammalian eIF3. One of the main structural domains shared by several eIF3 subunits -the PCI (for Proteasome, COP9, Initiation factor 3) domain -is shown in bold in both panels; adopted from (Zeman et al, 2019). (C, D) Schematic representation of the arrangement of eIF3 subunits in the two available conformations that are deduced from the available Cryo-EM analysis (adapted from ).…”

“…The eIF3 complex, composed of five subunits in yeast (a/Tif32, b/Prt1, c/Nip1, g/Tif35, i/Tif34) and twelve in mammals (a, b, c, d, e, f, g, h, i, k, l, m) (Figure S1A,B), is known to be critical for efficient progression of most of the initiation steps, yet its complete structure has not been determined from any organism (reviewed in (Cate, 2017;Valasek et al, 2017)). The assembly pathway for human and N. crassa (similar in composition to human) eIF3 was recently described (Smith et al, 2016;Wagner et al, 2014;Wagner et al, 2016) but it remains to be examined for the most extensively studied S. cerevisiae eIF3 complex (Zeman et al, 2019). Recent structural studies of various PICs revealed several well-resolved but otherwise discontinuous densities attributed to various eIF3 modules that together nearly embrace the entire 40S.…”

Structural inventory of native ribosomal ABCE1-43S pre-initiation complexes

“…In yeast, the PCI-MPN core consists of the two subunits eIF3a (Rpg1/Tif32) and eIF3c (Nip1), whereas in mammals, it is formed by an octamer of eIFs 3a, 3c, 3e, 3f, 3h, 3i, 3k and 3l (Valasek et al, 2017). The peripheral subunits consist of the socalled yeast-like core (YLC) module, containing eIF3b (Prt1), eIF3g (Tif35), and eIF3i (Tif34), as well as the C-terminus of eIF3a, the N-terminal domain of eIF3c that interacts with eIF1 and eIF5 (Valasek et al, 2003;Valasek et al, 2004;Yamamoto et al, 2005;Zeman et al, 2019), and in mammals eIF3d. In addition, eIF3j is associated with eIF3 but does not belong to its core, and plays a special role (Block et al, 1998;Valasek et al, 1999).…”