Yaser Hashem

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs 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 mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5, 6, 7, 9, 10, 11, 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 eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, 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 complex, 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 favouring translation of viral mRNAs.

High-Resolution Cryo-Electron Microscopy Structure of the Trypanosoma Brucei Ribosome.

Ribosomes, the protein factories of 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 ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. 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 of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.

Structure of mammalian eif3 in the context of the 43s preinitiation complex.

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5′-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.

Integrity of the P-site is probed during maturation of the 60S ribosomal subunit

The P-site of the 60S ribosomal subunit signals to Tif6 via Elf1 during ribosomal maturation, suggesting a quasifunctional check of the integrity of the 60S subunit before the first round of translation.

Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.

The ABC-F protein EttA gates ribosome entry into the translation elongation cycle

ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.

Visualizing the transfer‐messenger RNA as the ribosome resumes translation

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer‐messenger RNA (tmRNA), a dual‐function molecule that contains a tRNA‐like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo‐electron microscopy, we obtained the first structure of an in vivo‐formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable ‘arc and fork’ structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.

EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics

Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, energy-dependent translational throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA–binding site. Using single-molecule fluorescence resonance energy transfer, we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, in which the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.

Dynamical features of the Plasmodium falciparum ribosome during translation.

Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials.

Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF

In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress‐induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation‐promoting factor (SaHPF) that we solved using cryo‐electron microscopy. Our reconstructions reveal that the N‐terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C‐terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD‐dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA. We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.