Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

Abstract

SummaryTranslation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

Eukaryotic aspects of translation initiation brought into focus

“…This implies that eIF5 would make only secondary contacts with the 40S subunit and therefore be relatively disordered with respect to it. The eIF3big complex has been weakly resolved in contact with eIF2 on the subunit interface in the presence of mRNA, a position that is considerably different compared with its position on the 40S solvent-exposed surface in complexes in the absence of mRNA, suggesting a conformational change during scanning [9,21]. While there is currently no mechanistic explanation for this observation, it appears plausible that might be involved in the communication of the stable incorporation of the ternary complex and mRNA to other factors bound by the scaffold ( figure 3). rstb.royalsocietypublishing.org Phil.…”

“…eIF4 is known to bind both the cap and, through poly-A binding protein, the tail of the translated mRNA, a pattern suspected to result in the proximity of the translational termination and start sites with obvious consequences for the efficiency of recycling of terminating ribosomes [11]; however, its exact structural arrangement with respect to the 40S remains one of the gaping holes in our knowledge. With the advent of higher-resolution cryo-electron microscopy (EM), it has proved possible, however, to obtain a relatively complete intermediate-resolution description of the organization of eIF3 from both yeast [8,9,13] and mammals [14][15][16] (figure 2). eIF3 circumnavigates the entire 40S ribosomal subunit [8,13], recruiting and coordinating essentially every element of the translational apparatus.…”

“…The eIF3big sub-complex is connected to the core PCI/MPN subcomplex through the extended a-helical carboxy-terminal tail of eIF3a. In mammals this region remains unresolved [14]; however, in yeast it has been observed bound across the solvent-exposed surface of the 40S, presumably contributing to the interaction surface with which eIF3 binds the 40S [8,9,13] and rabbit [14 -16] eIF3 within the context of the 40S initiation complexes are shown as stylized models from the solvent-exposed face of the complex. Representations of the peripheral helicase, DHX29, within the rabbit complex, and eIF3j, within the yeast complex, are shown for context, although it is important to note that, in these two cases, it has not yet proved possible to generate a molecular interpretation.…”

“…Binding by eIF1A keeps the rRNA bases within the decoding site in a flippedout conformation, facilitating mRNA scanning by minimizing the interaction between the mRNA and rRNA in the mRNA channel [18], and eIF1 protrudes with a conserved loop into the mRNA channel, ready to sense cognate codon-anticodon base pairing [17,19]. The ternary complex, which consists of charged Met-tRNAi in a selective complex in which the acceptor stem is cradled by eIF2, is incorporated directly into the P-site on its recruitment by the multifactor complex [9,14,15,20]. eIF2 is a GTPase, which binds in a state in which GTP has already been hydrolysed.…”

“…The only significant contact is made with the head within the E-site adjacent to the P-site occupied by the tRNA itself [20]. This ensures that the ternary complex moves with the flexible 40S head in relation to the body of the subunit, while the contacts made with eIF1 and eIF1A will vary with head movement [9]. The eIF2 GAP eIF5 was resolved only extremely weakly as a cloud of density abutting eIF2 [20].…”