The ribosome plays a central role in translating the genetic code into amino acid sequences during polypeptide synthesis. In each cycle of peptide elongation, the ribosome discriminates between correct and incorrect aminoacyl-tRNAs based on the codon present in its A-site. To ensure high fidelity, ribosomes employ multiple proofreading mechanisms that reduce the selection of incorrect aminoacyl-tRNAs. Initial proofreading of incorrect tRNAs (near-cognate or non-cognate) is well understood in prokaryotic ribosomes but incompletely understood in eukaryotic systems. To investigate tRNA selection and accommodation for both cognate and near-cognate tRNAs, we employed single-molecule fluorescence resonance energy transfer with an in vitro eukaryotic translation system assembled on an error-prone mRNA sequence. We compared tRNA binding and accommodation rates of tryptophan-aminoacyl-tRNA in the ribosomal A-site containing either cognate or near-cognate codons. Although initial sampling of near-cognate tRNAs was slower than for cognate tRNA, subsequent near-cognate sampling events proceeded more rapidly than the initial near-cognate event. Increasing the concentration of near-cognate aminoacyl-tRNA surprisingly decreased its accommodation efficiency, a phenomenon not seen with cognate tRNAs. These findings suggest that rejection of a near-cognate tRNA induces an altered ribosomal conformation that enhances discrimination against further errors while simultaneously accelerating the tRNA sampling rate.

Premature termination codons in mRNAs result from nonsense mutations and hinder the translation of full-length, functional proteins. Nonsense mutations cause numerous serious genetic diseases, including cystic fibrosis and Duchenne muscular dystrophy. Several small-molecule drugs have been reported that could potentially ameliorate these diseases by promoting translational readthrough at the premature termination codon. However, utilization of many of these molecules faces problems such as limited efficacy or high cellular toxicity. Using a selection strategy in Saccharomyces cerevisiae coupling suppression of endogenous nonsense mutations to cell survival, we identified ten readthrough-promoting cyclic peptides from a DNA-encoded library. The selected cyclic peptides suppress nonsense mutations in various reporter genes, and the candidates inducing the highest readthrough levels display no observable cytotoxicity in yeast. Mutational analysis of the most promising cyclic peptide demonstrate that most amino acid side chains contribute to the readthrough-stimulating activity. Importantly, this cyclic peptide appears to bind directly to the eukaryotic core translation machinery and promotes readthrough in vitro by interfering with ribosomal decoding. Our results suggest that small, cyclic peptides selected in vivo could represent a novel drug type to treat the many incurable human genetic diseases that are caused by nonsense mutations. ### Competing Interest Statement A university-owned patent application (with C.R.K. and N.B. as inventors) of the identified readthrough-promoting cyclic peptides has been submitted. Novo Nordisk Foundation, https://ror.org/04txyc737, Tømrermester Jørgen Holm og Hustru Elisa f. Hansens Mindelegat, , Fabrikant Einar Willumsens Mindelegat, , National Institutes of Health, , R35GM118139

Premature termination codon (PTC) diseases account for ∼12% of all human disease mutations. Although there are no FDA approved treatments for increasing PTC readthrough, one readthrough inducing drug, ataluren, has conditional approval for treatment of Duchenne muscular dystrophy elsewhere. Ataluren displays low toxicity in clinical trials for treatment of PTC diseases, but its therapeutic effects are inconsistent. The messenger RNA (mRNA) sequence context of a PTC is a major determinant of PTC readthrough efficiency. We have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC) catalysis of translation termination. Here, using an in vitro reconstituted system, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate RFC activity in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results suggest a potential rationale for the variability of ataluren effectiveness in stimulating readthrough. We hypothesize that patients harboring a PTC mutation within a sequence context promoting strong interaction with RFC will be resistant to ataluren, but that ataluren treatment will be more effective for patient sequences conferring weaker interaction with RFC.

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in a patient's DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent. The identity of the stop codon and its sequence context are major determinants of PTC readthrough efficiency in both the absence and presence of nonsense suppressors. Previously we have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC, eRF1.eRF3.GTP)-dependent catalysis of translation termination. Here, using an in vitro reconstituted system (PURE-LITE) and both ensemble and single molecule assays, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate the catalytic activity of RFC in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, each of which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results provide an attractive rationale for the variability of ataluren effectiveness in stimulating readthrough in clinical trials. Patients harboring a PTC mutation with a sequence context promoting strong interaction with RFC are predicted to be resistant to ataluren, whereas ataluren treatment should be more effective for patient sequences conferring weaker interaction with RFC.

Mutations resulting in premature stop codons (PSCs) or nonsense mutations, are the root cause of >1,000 genetic diseases. However, the factors determining PSC suppression efficiency remain elusive. Using a reconstituted eukaryotic translation system, we demonstrate that release factor complex (RFC, eRF1.eRF3.GTP) dependent termination activities at all three nonsense codons are strongly influenced by downstream sequences found in cystic fibrosis and Marfan syndrome patient mRNA sequences, with variation among EC50RFC values exceeding 100-fold.

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with anin vitroeukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found that, following an initial slow sampling event, subsequent near-cognate sampling events proceeded more rapidly than the initial event. Further, we found a strong negative correlation between the concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to formation of an altered ribosomal conformation that assists in rejecting subsequent incorrect tRNA interactions.

Premature stop codons (PSCs) arrest translation of full-length protein and trigger nonsense-mediated decay (NMD) of mRNA. Nonsense suppression by translational readthrough inducing drugs (TRIDs) like ataluren can restore protein function by inhibiting translation termination at PSCs catalyzed by release factor complex (RFC, eRF1.eRF3.GTP). We seek to understand what determines readthrough efficiency and apply our results to improve treatment of PSC diseases such as cystic fibrosis (CF). For this purpose, we utilize an in vitro translation system to examine the effects on RFC enzymatic activity of varying PSCs and downstream mRNA sequences in pretermination complexes, as measured with a high throughput fluorescence anisotropy assay and single-molecule TIRF microscopy. We have so far studied a total of seven sequences downstream from stop codons. Within this limited set, we find much lower EC50RFC values for sequences with stop codons UAG or UAA than for sequences having a UGA stop codon. These sequences display a ∼170-fold difference in measured EC50RFC values, a much larger range than the very limited range of RFC binding rates, 1.7-fold. We speculate that this difference is due, at least in part, to differences in the rate of reversible RFC dissociation. Additional sequences are currently under study. Our preliminary results explain, at least in part, why readthrough frequency is generally much higher for the UGA stop codon than for the UAG or UAA stop codons. They further suggest that combinations of drugs acting orthogonally to stimulate readthrough may be required to treat some PSC diseases, and that consideration of the mRNA sequence context flanking a nonsense codon might be critical for developing patient-specific therapeutic regimens.

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation.

Many genetic disorders are caused by premature stop codon (PSC) mutations, but only one TRID (Translational readthrough-inducing drug), ataluren, has been approved for clinical use. Recently, we used single-molecule TIRF with a cell-free in vitro assay and found that ataluren competitively inhibits productive release factor complex (RFC, eRF1.eRF3.GTP) binding to the pre-termination complex. We found that such inhibition occurs before or at the peptidyl-tRNA hydrolysis step (Huang et al., Nat. Comm., 2022, 13: 2413). Here we report new results using an sm-FRET assay with a Cy3-labeled peptidyl-tRNA bound in the ribosomal P-site adjacent to a UGA stop codon in the A-site and Cy5-labeled human eRF1. Upon RFC binding, we observe transient FRET efficiency, E = ∼0.25, between Cy5-eRF1 and Cy3-tRNA, consistent with the successful accommodation of eRF1 within the A-site. Following peptidyl-tRNA hydrolysis, we find a strong correlation between eRF1 and tRNA dissociation times, unlike the weak correlation between peptide release and tRNA dissociation times that we observed previously (Huang et al., ibid.). Additionally, we found that ataluren and added near-cognate suppressor tRNA each had a significant inhibitory effect on the arrival time of RFCs at the ribosome, and that ataluren in combination with the aminoglycoside G418 had an even stronger inhibitory effect compared with ataluren or G418 alone. These results support our earlier suggestion (Ng et al., PNAS, 2021, 118: e2020599118) that ataluren or ataluren-like TRIDs could potentiate the therapeutic effects of aminoglycosides on PSC diseases. We expect that our ongoing studies will aid in elucidating the readthrough mechanisms of other TRIDs leading to enhanced and safer treatments of PSC diseases.

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren's mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren's binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren's low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.

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.

Genetic diseases are often caused by nonsense mutations, but only one TRID (translation readthrough inducing drug), ataluren, has been approved for clinical use. Ataluren inhibits release factor complex (RFC) termination activity, while not affecting productive binding of near-cognate ternary complex (TC, aa-tRNA.eEF1A.GTP). Here we use photoaffinity labeling to identify two sites of ataluren binding within rRNA, proximal to the decoding center (DC) and the peptidyl transfer center (PTC) of the ribosome, which are directly responsible for ataluren inhibition of termination activity. A third site, within the RFC, has as yet unclear functional consequences. Using single molecule and ensemble fluorescence assays we also demonstrate that termination proceeds via rapid RFC-dependent hydrolysis of peptidyl-tRNA followed by slow release of peptide and tRNA from the ribosome. Ataluren is an apparent competitive inhibitor of productive RFC binding, acting at or before the hydrolysis step. We propose that designing more potent TRIDs which retain ataluren’s low toxicity should target areas of the RFC binding site proximal to the DC and PTC which do not overlap the TC binding site.

Several novel potential therapies directed towards the treatment of premature stop codon (PSC) diseases involve use of therapeutic agents called translation readthrough inducing drugs (TRIDs). As yet, only one TRID, ataluren, has been approved for clinical use. By using an in vitro eukaryotic PURE-LITE system with the Cricket Paralysis Virus-Internal Ribosome Entry Site, we recently demonstrated that ataluren promotes readthrough of a nonsense codon exclusively via inhibition of termination activity by the release factor complex (RFC, eRF1.eRF3.GTP).

The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo.

The polypeptide elongation cycle is much less studied and understood in eukaryotes than in prokaryotes. We are investigating eukaryotic elongation by single molecule FRET using fluorescently labeled tRNAs (fl-tRNAs) and ribosomes programmed with cricket paralysis IRES attached to an mRNA sequence. Stalled pre-translocation (PRE) complexes with fl-tRNAs occupying the A- and P-sites, primarily exhibit one low-FRET state at 5 mM Mg2+ but fluctuate between a low-FRET and high-FRET state when the concentration of Mg2+ is raised to 10 mM and 15 mM. In contrast, in stalled bacterial PRE complexes, A- and P-site tRNAs fluctuate between a high-FRET classical state and a low-FRET hybrid state at all three Mg2+ concentrations. Addition of eEF2•GTP to the eukaryotic PRE complex results in an increase in tRNA-tRNA FRET efficiency from E = 0.27 to E = 0.55, consistent with translocation of the tRNAs into the P- and E-sites of a post-translocation (POST) complex. The distribution of times between exposure to eEF2 and this FRET increase exhibits a concentration-dependent lag phase and exponential completion that are not well described by two successive first-order processes, suggesting that several intervening steps occur between eEF2 binding and translocation. Following the putative translocation step, the E-site tRNA remains bound for at least 0.2 seconds, again suggesting that one or more further step(s) occur before E-site tRNA departure. Our single-molecule FRET studies provide mechanistic insights into the eukaryotic protein synthesis elongation cycle and suggest that the translocation step differs qualitatively between eukaryotic and prokaryotic systems.