Rate-limiting hydrolysis in ribosomal release reactions revealed by ester activation.

Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect. Here, we tested this by activating the ester electrophile at the Escherichia coli ribosomal P site using a trifluorine-substituted amino acid. Quench-flow kinetics revealed that RF1-catalyzed release could be accelerated, but only at pH 6.2-7.7 and not higher pH. This provided direct evidence for rate-limiting hydrolysis at physiological or lower pH and a different rate limitation at higher pH. Additionally, we optimized RF-free release catalyzed by unacylated tRNA or the CCA trinucleotide (in 30% acetone). We determined that these two model release reactions, although very slow, were surprisingly accelerated by the trifluorine analog but to a different extent from each other and from RF-catalyzed release. Hence, hydrolysis was rate limiting in all three reactions. Furthermore, in 20% ethanol, we found that there was significant competition between fMet-ethyl ester formation and release in all three release reactions. We thus favor proposed mechanisms for translation termination that do not require a fully-negatively-charged OH- nucleophile.

Structural basis of translation termination, rescue, and recycling in mammalian mitochondria.

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.

Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination.

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a "leaky" stop codon context, which likely defines the basis of nonsense suppression.

Translational coupling via termination-reinitiation in archaea and bacteria.

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.

The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy.

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

A dual role of the ribosome-bound chaperones RAC/Ssb in maintaining the fidelity of translation termination.

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.

Robustness by intrinsically disordered C-termini and translational readthrough.

During protein synthesis genetic instructions are passed from DNA via mRNA to the ribosome to assemble a protein chain. Occasionally, stop codons in the mRNA are bypassed and translation continues into the untranslated region (3'-UTR). This process, called translational readthrough (TR), yields a protein chain that becomes longer than would be predicted from the DNA sequence alone. Protein sequences vary in propensity for translational errors, which may yield evolutionary constraints by limiting evolutionary paths. Here we investigated TR in Saccharomyces cerevisiae by analysing ribosome profiling data. We clustered proteins as either prone or non-prone to TR, and conducted comparative analyses. We find that a relatively high frequency (5%) of genes undergo TR, including ribosomal subunit proteins. Our main finding is that proteins undergoing TR are highly expressed and have intrinsically disordered C-termini. We suggest that highly expressed proteins may compensate for the deleterious effects of TR by having intrinsically disordered C-termini, which may provide conformational flexibility but without distorting native function. Moreover, we discuss whether minimizing deleterious effects of TR is also enabling exploration of the phenotypic landscape of protein isoforms.

Operative Binding of Class I Release Factors and YaeJ Stabilizes the Ribosome in the Nonrotated State.

During translation, the small subunit of the ribosome rotates with respect to the large subunit primarily between two states as mRNA is being translated into a protein. At the termination of bacterial translation, class I release factors (RFs) bind to a stop codon in the A-site and catalyze the release of the peptide chain from the ribosome. Periodically, mRNA is truncated prematurely, and the translating ribosome stalls at the end of the mRNA forming a nonstop complex requiring one of several ribosome rescue factors to intervene. One factor, YaeJ, is structurally homologous with the catalytic region of RFs but differs by binding to the ribosome directly through its C-terminal tail. Structures of the ribosome show that the ribosome adopts the nonrotated state conformation when these factors are bound. However, these studies do not elucidate the influence of binding to cognate or noncognate codons on the dynamics of intersubunit rotation. Here, we investigate the effects of wild-type and mutant forms of RF1, RF2, and YaeJ binding on ribosome intersubunit rotation using single-molecule Förster resonance energy transfer. We show that both RF1 binding and RF2 binding are sufficient to shift the population of posthydrolysis ribosome complexes from primarily the rotated to the nonrotated state only when a cognate stop codon is present in the A-site. Similarly, YaeJ binding stabilizes nonstop ribosomal complexes in the nonrotated state. Along with previous studies, these results are consistent with the idea that directed conformational changes and binding of subsequent factors to the ribosome are requisite for efficient termination and ribosome recycling.

The Differences in Local Translatome across Distinct Neuron Types Is Mediated by Both Baseline Cellular Differences and Post-transcriptional Mechanisms.

Local translation in neurites is a phenomenon that enhances the spatial segregation of proteins and their functions away from the cell body, yet it is unclear how local translation varies across neuronal cell types. Further, it is unclear whether differences in local translation across cell types simply reflect differences in transcription or whether there is also a cell type-specific post-transcriptional regulation of the location and translation of specific mRNAs. Most of the mRNAs discovered as being locally translated have been identified from hippocampal neurons because their laminar organization facilitates neurite-specific dissection and microscopy methods. Given the diversity of neurons across the brain, studies have not yet analyzed how locally translated mRNAs differ across cell types. Here, we used the SynapTRAP method to harvest two broad cell types in the mouse forebrain: GABAergic neurons and layer 5 projection neurons. While some transcripts overlap, the majority of the local translatome is not shared across these cell types. In addition to differences driven by baseline expression levels, some transcripts also exhibit cell type-specific post-transcriptional regulation. Finally, we provide evidence that GABAergic neurons specifically localize mRNAs for peptide neurotransmitters, including somatostatin and cortistatin, suggesting localized production of these key signaling molecules in the neurites of GABAergic neurons. Overall, this work suggests that differences in local translation in neurites across neuronal cell types are poised to contribute substantially to the heterogeneity in neuronal phenotypes.

Dynamic basis of fidelity and speed in translation: Coordinated multistep mechanisms of elongation and termination.

As the universal machine that transfers genetic information from RNA to protein, the ribosome synthesizes proteins with remarkably high fidelity and speed. This is a result of the accurate and efficient decoding of mRNA codons via multistep mechanisms during elongation and termination stages of translation. These mechanisms control how the correct sense codon is recognized by a tRNA for peptide elongation, how the next codon is presented to the decoding center without change of frame during translocation, and how the stop codon is discriminated for timely release of the nascent peptide. These processes occur efficiently through coupling of chemical energy expenditure, ligand interactions, and conformational changes. Understanding this coupling in detail required integration of many techniques that were developed in the past two decades. This multidisciplinary approach has revealed the dynamic nature of translational control and uncovered how external cellular factors such as tRNA abundance and mRNA modifications affect the synthesis of the protein product. Insights from these studies will aid synthetic biology and therapeutic approaches to translation.

The ribosome-bound quality control complex remains associated to aberrant peptides during their proteasomal targeting and interacts with Tom1 to limit protein aggregation.

Protein quality control mechanisms eliminate defective polypeptides to ensure proteostasis and to avoid the toxicity of protein aggregates. In eukaryotes, the ribosome-bound quality control (RQC) complex detects aberrant nascent peptides that remain stalled in 60S ribosomal particles due to a dysfunction in translation termination. The RQC complex polyubiquitylates aberrant polypeptides and recruits a Cdc48 hexamer to extract them from 60S particles in order to escort them to the proteasome for degradation. Whereas the steps from stalled 60S recognition to aberrant peptide polyubiquitylation by the RQC complex have been described, the mechanism leading to proteasomal degradation of these defective translation products remains unknown. We show here that the RQC complex also exists as a ribosome-unbound complex during the escort of aberrant peptides to the proteasome. In addition, we identify a new partner of this light version of the RQC complex, the E3 ubiquitin ligase Tom1. Tom1 interacts with aberrant nascent peptides and is essential to limit their accumulation and aggregation in the absence of Rqc1; however, its E3 ubiquitin ligase activity is not required. Taken together, these results reveal new roles for Tom1 in protein quality control, aggregate prevention, and, therefore, proteostasis maintenance.

RNA helicase DDX19 stabilizes ribosomal elongation and termination complexes.

The human DEAD-box RNA-helicase DDX19 functions in mRNA export through the nuclear pore complex. The yeast homolog of this protein, Dbp5, has been reported to participate in translation termination. Using a reconstituted mammalian in vitro translation system, we show that the human protein DDX19 is also important for translation termination. It is associated with the fraction of translating ribosomes. We show that DDX19 interacts with pre-termination complexes (preTCs) in a nucleotide-dependent manner. Furthermore, DDX19 increases the efficiency of termination complex (TC) formation and the peptide release in the presence of eukaryotic release factors. Using the eRF1(AGQ) mutant protein or a non-hydrolysable analog of GTP to inhibit subsequent peptidyl-tRNA hydrolysis, we reveal that the activation of translation termination by DDX19 occurs during the stop codon recognition. This activation is a result of DDX19 binding to preTC and a concomitant stabilization of terminating ribosomes. Moreover, we show that DDX19 stabilizes ribosome complexes with translation elongation factors eEF1 and eEF2. Taken together, our findings reveal that the human RNA helicase DDX19 actively participates in protein biosynthesis.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

Mechanism, factors, and physiological role of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent, multistep process that degrades irregular or faulty messenger RNAs (mRNAs). NMD mainly targets mRNAs with a truncated open reading frame (ORF) due to premature termination codons (PTCs). In addition, NMD also regulates the expression of different types of endogenous mRNA substrates. A multitude of factors are involved in the tight regulation of the NMD mechanism. In this review, we focus on the molecular mechanism of mammalian NMD. Based on the published data, we discuss the involvement of translation termination in NMD initiation. Furthermore, we provide a detailed overview of the core NMD machinery, as well as several peripheral NMD factors, and discuss their function. Finally, we present an overview of diseases associated with NMD factor mutations and summarize the current state of treatment for genetic disorders caused by nonsense mutations.

ABCE1 is a highly conserved RNA silencing suppressor.

ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

Distinct roles for release factor 1 and release factor 2 in translational quality control.

In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.

Resolving nonstop translation complexes is a matter of life or death.

Problems during gene expression can result in a ribosome that has translated to the 3' end of an mRNA without terminating at a stop codon, forming a nonstop translation complex. The nonstop translation complex contains a ribosome with the mRNA and peptidyl-tRNA engaged, but because there is no codon in the A site, the ribosome cannot elongate or terminate the nascent chain. Recent work has illuminated the importance of resolving these nonstop complexes in bacteria. Transfer-messenger RNA (tmRNA)-SmpB specifically recognizes and resolves nonstop translation complexes in a reaction known as trans-translation. trans-Translation releases the ribosome and promotes degradation of the incomplete nascent polypeptide and problematic mRNA. tmRNA and SmpB have been found in all bacteria and are essential in some species. However, other bacteria can live without trans-translation because they have one of the alternative release factors, ArfA or ArfB. ArfA recruits RF2 to nonstop translation complexes to promote hydrolysis of the peptidyl-tRNAs. ArfB recognizes nonstop translation complexes in a manner similar to tmRNA-SmpB recognition and directly hydrolyzes the peptidyl-tRNAs to release the stalled ribosomes. Genetic studies indicate that most or all species require at least one mechanism to resolve nonstop translation complexes. Consistent with such a requirement, small molecules that inhibit resolution of nonstop translation complexes have broad-spectrum antibacterial activity. These results suggest that resolving nonstop translation complexes is a matter of life or death for bacteria.

Structural and functional insights into Saccharomyces cerevisiae Tpa1, a putative prolylhydroxylase influencing translation termination and transcription.

Efficiency of translation termination relies on the specific recognition of the three stop codons by the eukaryotic translation termination factor eRF1. To date only a few proteins are known to be involved in translation termination in eukaryotes. Saccharomyces cerevisiae Tpa1, a largely conserved but uncharacterized protein, has been described to associate with a messenger ribonucleoprotein complex located at the 3' end of mRNAs that contains at least eRF1, eRF3, and Pab1. Deletion of the TPA1 gene results in a decrease of translation termination efficacy and an increase in mRNAs half-lives and longer mRNA poly(A) tails. In parallel, Schizosaccharomyces pombe Ofd1, a Tpa1 ortholog, and its partner Nro1 have been implicated in the regulation of the stability of a transcription factor that regulates genes essential for the cell response to hypoxia. To gain insight into Tpa1/Ofd1 function, we have solved the crystal structure of S. cerevisiae Tpa1 protein. This protein is composed of two equivalent domains with the double-stranded ß-helix fold. The N-terminal domain displays a highly conserved active site with strong similarities with prolyl-4-hydroxylases. Further functional studies show that the integrity of Tpa1 active site as well as the presence of Yor051c/Ett1 (the S. cerevisiae Nro1 ortholog) are essential for correct translation termination. In parallel, we show that Tpa1 represses the expression of genes regulated by Hap1, a transcription factor involved in the response to levels of heme and oxygen. Altogether, our results support that Tpa1 is a putative enzyme acting as an oxygen sensor and influencing several distinct regulatory pathways.

Terminating human mitochondrial protein synthesis: a shift in our thinking.

Until recently, human mitochondria were regarded as unusual as they appeared to employ four stop codons to terminate translation. In addition to the UAA/UAG of the universal genetic code, two arginine triplets (AGA/AGG) had been re-assigned as termination signals. This posed the conundrum of what factor was responsible for recognizing these triplets to promote translation termination? Recent data indicates that in fact no protein is required to recognize AGA/AGG. Indeed, it is the absence of any cognate factor, tRNA or polypeptide that is important. On encountering either of these 'hungry' codons at the end of an open reading frame, instead of requiring a novel or modified release factor, human mitoribosomes employ -1 frameshifting to reposition a standard UAG codon in the A-site, indicating that only the universal UAA and UAG are used as stop codons. This renders a single mitochondrial release factor, mtRF1a, previously shown to be capable of terminating 11 of the 13 open reading frames encoded by the mitochondrial genome, to be sufficient to release all nascent human mitochondrial gene products from the mitoribosome.