Markus Pech

Structure of the native Sec61 protein-conducting channel.

In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment.

The Crystal Structure of Archaeal Nascent Polypeptide-associated Complex (NAC) Reveals a Unique Fold and the Presence of a Ubiquitin-associated Domain

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is highly conserved from yeast to humans. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila, and Caenorhabditis elegans. NAC was suggested to protect the nascent chain from inappropriate early interactions with cytosolic factors. Eukaryotic NAC is a heterodimer with two subunits sharing substantial homology with each other. All sequenced archaebacterial genomes exhibit only one gene homologous to the NAC subunits. Here we present the first archaebacterial NAC homolog. It forms a homodimer, and as eukaryotic NAC it is associated with ribosomes and contacts the emerging nascent chain on the ribosome. We present the first crystal structure of a NAC protein revealing two structural features: (i) a novel unique protein fold that mediates dimerization of the complex, and (ii) a ubiquitin-associated domain that suggests a yet unidentified role for NAC in the cellular protein quality control system via the ubiquitination pathway. Based on the presented structure we propose a model for the eukaryotic heterodimeric NAC domain.

The complex of tmRNA-SmpB and EF-G on translocating ribosomes.

Bacterial ribosomes stalled at the 3′ end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD–SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome–tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S–tmRNA–SmpB–EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TIPOST) presents the TLD–SmpB module in an intrasubunit ap/P hybrid site and a tRNAfMet in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA–SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations

Elongation factor 4 (EF4) is one of the most conserved proteins present in bacteria as well as in mitochondria and chloroplasts of eukaryotes. Although EF4 has the unique ability to catalyze the back-translocation reaction on posttranslocation state ribosomes, the physiological role of EF4 remains unclear. Here we demonstrate that EF4 is stored at the membrane of Escherichia coli cells and released into the cytoplasm upon conditions of high ionic strength or low temperature. Under such conditions, wild-type E. coli cells overgrow mutant cells lacking the EF4 gene within 5–10 generations. Elevated intracellular Mg2+ concentrations or low temperature retard bacterial growth and inhibit protein synthesis, probably because of formation of aberrant elongating ribosomal states. We suggest that EF4 binds to these stuck ribosomes and remobilizes them, consistent with the EF4-dependent enhancement (fivefold) in protein synthesis observed under these unfavorable conditions. The strong selective advantage conferred by the presence of EF4 at high intracellular ionic strength or low temperatures explains the ubiquitous distribution and high conservation of EF4.

RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.

NOA1 is an essential GTPase required for mitochondrial protein synthesis

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate binding protein that localizes predominantly to mitochondria in mammalian cells. Here we determine NOA1 function through generation of knock-out mice and in vitro assays.

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Significance Until now, two initiation modes for bacterial translation have been described: (i) the standard 30S-binding mode, where the small ribosomal subunit selects the initiation site on an mRNA with the help of three initiation factors (IFs), and (ii) the rare initiation of leaderless mRNAs, which are mRNAs carrying the initiation AUG within the first 5 nt at the 5′-end. The existence of a third “70S-scanning” mode for bacterial initiation was conjectured in past decades but has remained experimentally unproven. Here, we demonstrate the existence of a 70S-scanning mode of initiation and characterize its mechanistic features. The three initiation modes demonstrate specific patterns of requirements for IF1 and IF3. According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1–IF3). Here, we describe a novel type of initiation termed “70S-scanning initiation,” where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine–Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.

Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.

Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome.

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.

The cryo-EM structure of a ribosome–Ski2-Ski3-Ski8 helicase complex

Getting rid of faulty mRNA The cell monitors the health of its mRNAs, destroying those that are faulty or damaged. Destruction by the exosome complex prevents them from being used to synthesize deranged and potentially dangerous proteins. Schmidt et al. determined the structure of the Ski helicase complex, which guides RNAs to the exosome complex destruction machinery in association with a mRNAbound ribosome. The end of the mRNA is threaded from the ribosome into the heart of the helicase, whence the message would be channeled into the maw of the exosome complex. Science, this issue p. 1431 The structure of a ribosome with messenger RNA (mRNA) bound to the Ski helicase complex reveals a step in the destruction of aberrant mRNA. Ski2-Ski3-Ski8 (Ski) is a helicase complex functioning with the RNA-degrading exosome to mediate the 3′-5′ messenger RNA (mRNA) decay in turnover and quality-control pathways. We report that the Ski complex directly associates with 80S ribosomes presenting a short mRNA 3′ overhang. We determined the structure of an endogenous ribosome-Ski complex using cryo–electron microscopy (EM) with a local resolution of the Ski complex ranging from 4 angstroms (Å) in the core to about 10 Å for intrinsically flexible regions. Ribosome binding displaces the autoinhibitory domain of the Ski2 helicase, positioning it in an open conformation near the ribosomal mRNA entry tunnel. We observe that the mRNA 3′ overhang is threaded directly from the small ribosomal subunit to the helicase channel of Ski2, primed for ongoing exosome-mediated 3′-5′ degradation.