Structural basis of TACO1-mediated efficient mitochondrial translation

Translation elongation is a universally conserved step in protein synthesis, relying on elongation factors that engage the ribosomal L7/L12 stalk to mediate aminoacyl-tRNA delivery, accommodation, and ribosomal translocation. Using in organello cryo-electron microscopy, we reveal how the mitochondrial translation accelerator TACO1 promotes efficient elongation on human mitoribosomes. TACO1 binds the mitoribosomal region typically bound by elongation factor Tu (mtEF-Tu), bridging the large and small subunits via contacts with 16S rRNA, bL12m, A-site tRNA, and uS12m. While active throughout elongation, TACO1 is especially critical when translating polyproline motifs. Its absence prolongs mtEF-Tu residence in A/T states, causes persistent mitoribosomal stalling and premature subunit dissociation. Structural analyses indicate that TACO1 competes with mtEF-Tu for mitoribosome binding, stabilizes A-site tRNA, and enhances peptidyl transfer through a mechanism distinct from EF-P and eIF5A. These findings suggest that bacterial TACO1 orthologs may serve analogous roles, highlighting an evolutionarily conserved strategy for maintaining elongation efficiency during challenging translation events.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

In addition to their function in protein synthesis, translating ribosomes serve as sensors that communicate the presence of aberrant messenger RNAs (mRNAs); however, how they recognize damage to their ribosomal RNA (rRNA) remains poorly understood. The conserved sarcin/ricin loop (SRL) of the 25S rRNA is a component of the GTPase center essential for ribosome movement during translation. In this study, we expressed an RNA N-glycosylase called pokeweed antiviral protein (PAP) in yeast Saccharomyces cerevisiae to specifically damage rRNA by hydrolysis of a purine base from the SRL. 25S rRNA depurination inhibited translation elongation, as shown by reduced incorporation of a methionine analog and binding of eukaryotic elongation factor 2 (eEF2) to ribosomes. PAP expression altered sucrose gradient profiles, increasing free subunits and 80S peaks and reducing polysomes without causing ribosome collisions. We discovered depurinated rRNA associated with 80S monosomes and polysomes, suggesting that cells would detect damage to rRNA during active translation. These ribosomes were ubiquitinated by E3 ligases Mag2 and Hel2, elements of the 18S nonfunctional rRNA decay (NRD) pathway involved in recognizing slow-moving ribosomes. Furthermore, mass spectrometry analysis revealed ubiquitination of ribosomal protein uS3, characteristic of 18S NRD. Even though the SRL is a component of the large ribosomal subunit, its depurination is signaled by ubiquitin ligases that recognize damage to the small subunit. We suggest that slow translation elongation is the factor that communicates SRL depurination to E3 ubiquitin ligases, which extends our understanding of how rRNA integrity is surveilled in yeast.

During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.

Translocation of mRNA and tRNAs through the ribosome is catalyzed by the universally conserved elongation factor (EF‐G in prokaryotes and EF‐2 in eukaryotes). It has been hypothesized that EF‐G undergoes structural rearrangements during translocation. Here, we test this hypothesis by following the movement of EF‐G relative to the small ribosomal subunit using single‐molecule Förster resonance energy transfer (smFRET). We show that ribosome‐bound EF‐G adopts distinct conformations corresponding to the pre‐ and post‐translocation states of the ribosome. Our results suggest that upon ribosomal translocation, domain IV of EF‐G, which is essential for the catalysis of translocation, moves toward the A site of the small ribosomal subunit and promotes translocation of peptidyl‐tRNA.Grant Funding Source: These studies were supported by grant from the US National Institute of Health no. GM‐099719

Protein synthesis is an intricate multi‐step process accomplished through a series of concerted actions of several different factors. Post initiation, translation elongation continues in a cyclic manner with the delivery of cognate aa‐tRNA to ribosomal A site, peptide bond formation and translocation to P‐site. The broad mechanism of action of elongation factors is known, however the regulation of translation elongation remains poorly understood. To safeguard timely and accurate gene expression, the protein synthesis machinery must be strictly regulated. Indeed the eukaryotic Elongation Factor 1A (eEF1A) shows extensive post‐translational modifications, both phosphorylation and methylation. In Saccharomyces cerevisiae, global proteomic analyses have reported at least 15 residues in eEF1A that are phosphorylated under different growth conditions. We have created a set of corresponding alanine (non‐phosphorylatable) and aspartate (phosphomimetic) mutations in these residues to study their phenotypic as well as enzymatic effects. Our in vivo and in vitro analyses in budding yeast show that S53D, Y416D and T430D mutant alleles are unable to support cell growth as the only form of eEF1A, while S18D, T259D and Y416A show slow growth and an initiation defect manifested by Gcn2 activation. Further experiments to test the translation fidelity, in vitro enzymatic activity and their ability to interact with cofactors are ongoing. In addition to translation elongation, eEF1A has been implicated in a wide variety of cellular processes, for example cytoskeleton organization through actin bundling, protein quality control, viral invasion and replication, etc. This work is aimed at identifying the events and factors that modulate eEF1A activity ensuring careful control of gene expression and appropriate cell function.Support or Funding InformationNIH grant RO1 GM057483

The factors involved in translation elongation are subject to sophisticated control mechanisms that come into play under a wide variety of conditions. Even though translation is most frequently controlled during the initiation phase (Chapter 1) and the regulatory mechanisms impinging on the initiation steps have received considerable attention (reviewed in several chapters of this book), accumulating information points to the elongation phase as a target for controls under defined circumstances. In this chapter, we focus on recent developments in understanding the control of elongation in mammalian cells. As a special case, we also discuss cotranslational protein targeting, a cellular process involving the control of elongation on an important class of mRNAs. REGULATION OF TRANSLATION ELONGATION The mechanism of peptide-chain elongation and the functions of translation elongation factors are described in Chapters 2 and 3. In addition, detailed aspects of the structure and function of eukaryotic elongation factor 2 (eEF2) are the subject of a recent informative review (Jorgensen et al. 2006). eEF2 is a phosphoprotein in mammalian cells, and most of the recent advances relate to the regulation of eEF2 and its cognate kinase, eEF2 kinase. eEF1A and eEF1B also are phosphoproteins and have been discussed in earlier reviews on this subject (Proud 2000; Traugh 2001; Browne and Proud 2002; Le Sourd et al. 2006). Significance of eEF2 Phosphorylation for the Control of Protein Synthesis Under a diverse range of conditions, the phosphorylation state of eEF2 changes in directions consistent with its having a role in regulating protein synthesis; i.e.,...

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.

Cardiac muscles of experimentally induced diabetic rats show a progressive decrease in the rate of protein synthesis. The decline in protein synthesis is associated with decreases in both the number and efficiency of cardiac ribosomes. In hearts from 48 h diabetic rats, the decrease in protein synthesis was accounted for solely by a 28% reduction in the ribosome content. In contrast, the inhibition of protein synthesis in hearts from 72 h diabetic rats resulted from a reduction in both the ribosome content (28%) and the translational efficiency (30%). The decreased translational efficiency was not associated with an increase of RNA in ribosomal subunits, indicating the defect resulted from an inhibition of peptide-chain elongation/termination. Diabetes of 72 h duration resulted in a 37% inhibition in the rate of peptide-chain elongation. The decreased rate of peptide-chain elongation was associated with a 66% reduction in the amount of elongation factor 2 (EF-2). Treatment of diabetic rats with insulin for 3 days was sufficient to reverse the effects of 72 h diabetes on protein synthesis, RNA content, and translational efficiency. Also, insulin therapy increased the EF-2 content of diabetic rats to control values. These studies suggest that decreased EF-2 content is a molecular mechanism for the impaired rates of peptide-chain elongation in diabetes.

eIF3 Associates with 80S Ribosomes to Promote Translation Elongation, Mitochondrial Homeostasis, and Muscle Health

A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled at the levels of both translation initiation and elongation. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step of peptide-chain elongation and is inhibited through phosphorylation by eEF2 kinase. In addition, p70S6 kinase can regulate protein synthesis by phosphorylating eEF2 kinase or via phosphorylation of ribosomal protein S6. We have recently shown that eEF2 kinase is also controlled by phosphorylation by AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. Moreover, the mammalian target of rapamycin has also been shown to be inhibited, indirectly, by AMPK, thus leading to the inhibition of p70S6 kinase. Although AMPK activation has been shown to modulate protein synthesis, it is unknown whether AMPK could also be a regulator of cardiac hypertrophic growth. Therefore, we investigated the role of AMPK activation in regulating protein synthesis during both phenylephrine- and Akt-induced cardiac hypertrophy. Metformin and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside were used to activate AMPK in neonatal rat cardiac myocytes. Activation of AMPK significantly decreased protein synthesis induced by phenylephrine treatment or by expression of constitutively active Akt. Activation of AMPK also resulted in decreased p70S6 kinase phosphorylation and increased phosphorylation of eEF2, suggesting that inhibition of protein synthesis involves the eEF2 kinase/eEF2 axis and/or the p70S6 kinase pathway. Together, our data suggest that the inhibition of protein synthesis by pharmacological activation of AMPK may be a key regulatory mechanism by which hypertrophic growth can be controlled.

Oxidative damage of 18S and 5S ribosomal RNA in digestive gland of mussels exposed to trace metals

Several microbial pathogens target host protein synthesis machinery, potentially limiting the innate immune responses of the host. In response, hosts trigger a defensive response, elevating immune gene transcripts. This counterintuitive response can have either beneficial or harmful effects on host survival. In this study, we conduct a comprehensive analysis of the impact of knocking down various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, inhibiting initiation and elongation factors has contrasting effects on C. elegans survival. Inhibiting translation initiation activates immune responses that protect the host from bacterial infection, while inhibiting translation elongation induces aberrant immune responses that, although clear the infection, are detrimental to the host. Our study reveals divergent roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection and identifies differential transcriptional reprogramming that could underlie these differences.