SUMMARY The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3′ UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.

A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation.DOI: http://dx.doi.org/10.7554/eLife.05534.001

During protein synthesis, ribosomes encounter many roadblocks, the outcomes of which are largely determined by substrate availability, amino acid features and reaction kinetics. Prolonged ribosome stalling is likely to be resolved by ribosome rescue or quality control pathways, whereas shorter stalling is likely to be resolved by ongoing productive translation. How ribosome function is affected by such hindrances can therefore have a profound impact on the translational output (yield) of a particular mRNA. In this Review, we focus on these roadblocks and the resumption of normal translation elongation rather than on alternative fates wherein the stalled ribosome triggers degradation of the mRNA and the incomplete protein product. We discuss the fundamental stages of the translation process in eukaryotes, from elongation through ribosome recycling, with particular attention to recent discoveries of the complexity of the genetic code and regulatory elements that control gene expression, including ribosome stalling during elongation, the role of mRNA context in translation termination and mechanisms of ribosome rescue that resemble recycling.

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1–3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4–6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that—compared with previous human NPC models obtained from purified NEs—the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation-inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non-translating 80S ribosomes stabilized by the 'clamping' Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34-Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34-Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34-Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.

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SUMMARY Protein synthesis plays an essential role in cell proliferation, differentiation and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, (-)-agelastatin A (AglA), possesses potent antitumor activity. Its underlying mechanism of action, however, has remained unknown. Using a systematic top-down approach, we show that AglA selectively inhibits protein synthesis. Using a high-throughput chemical footprinting method, we mapped the AglA-binding site to the ribosomal A site. A 3.5-Å crystal structure of the 80S eukaryotic ribosome from S. cerevisiae in complex with AglA was obtained, revealing multiple conformational changes of the nucleotide bases in the ribosome accompanying the binding of AglA. Together, these results have unraveled the mechanism of inhibition of eukaryotic translation by AglA at atomic level, paving the way for future structural modifications to develop AglA analogs into novel anticancer agents.