Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, Met-tRNAiMet, eukaryotic initiation factor 2 (eIF2) and GTP form a ternary complex (TC). The TC, eIF3, eIF1 and eIF1A cooperatively bind to the 40S subunit yielding the 43S preinitiation complex, which is ready to attach to mRNA and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α-subunit and supports Met-tRNAiMet in a novel P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.

Hepatitis C virus (HCV) and Classical swine fever virus (CSFV) mRNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5’-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs1. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit2–8, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound Met-tRNAiMet to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF32,5–7,9–12, but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF313 and the HCV IRES8 revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components13. Here, we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Strikingly, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S/IRES binary complex8, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favoring translation of viral mRNAs.

A Brownian machine, a tiny device buffeted by the random motions of molecules in the environment, is capable of exploiting these thermal motions for many of the conformational changes in its work cycle. Such machines are now thought to be ubiquitous, with the ribosome, a molecular machine responsible for protein synthesis, increasingly regarded as prototypical. Here we present a new analytical approach capable of determining the free-energy landscape and the continuous trajectories of molecular machines from a large number of snapshots obtained by cryogenic electron microscopy. We demonstrate this approach in the context of experimental cryogenic electron microscope images of a large ensemble of nontranslating ribosomes purified from yeast cells. The freeenergy landscape is seen to contain a closed path of low energy, along which the ribosome exhibits conformational changes known to be associated with the elongation cycle. Our approach allows model-free quantitative analysis of the degrees of freedom and the energy landscape underlying continuous conformational changes in nanomachines, including those important for biological function.cryo-electron microscopy | elongation cycle | manifold embedding | nanomachines | translation

Ribosomes, constituting protein factories in living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome1–6 reveal its increased complexity compared to its prokaryotic counterpart7–8, manifested mainly in the presence of eukaryotic-specific ribosomal proteins and additional rRNA insertions, called expansion segments (ES)9. These structures also point out some differences among species, in part represented by the size and arrangement of these ESs. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome from Trypanosoma brucei, the parasite transmitted by the Tse-tse fly and causing African sleeping sickness. Our atomic model reveals the unique features of this ribosome, characterized mainly by the presence of extraordinarily large ESs and ribosomal proteins extensions leading to the formation of four additional intersubunit bridges, and additional rRNA insertions including one large rRNA domain nonexistent in other eukaryotes. The kinetoplastid large ribosomal subunit (LSU) rRNA chain is uniquely cleaved into six pieces10–12; our structure reveals the five cleavage sites and suggests the importance of the cleavage for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA ESs for the translation regulation process. Our structure could serve as a basis for future experiments aiming at understanding the functional importance of these kinetoplastid-specific ribosomal features in the protein translation regulation, an essential step toward finding effective and safe kinetoplastid-specific drugs.

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