A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism

Ando, Yushin; Kobo, Akinao; Niwa, Tatsuya; Yamakawa, Ayako; Konoma, Suzuna; Kobayashi, Yuki; Nureki, Osamu; Taguchi, Hideki; Itoh, Yuzuru; Chadani, Yuhei

doi:10.1038/s41467-025-57659-z

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Published: 08 March 2025

A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism

Nature Communications volume 16, Article number: 2323 (2025) Cite this article

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Abstract

Protein synthesis by ribosomes produces functional proteins but also serves diverse regulatory functions, which depend on the coding amino acid sequences. Certain nascent peptides interact with the ribosome exit tunnel to arrest translation and modulate themselves or the expression of downstream genes. However, a comprehensive understanding of the mechanisms of such ribosome stalling and its regulation remains elusive. In this study, we systematically screen for unidentified ribosome arrest peptides through phenotypic evaluation, proteomics, and mass spectrometry analyses, leading to the discovery of the arrest peptides PepNL and NanCL in E. coli. Our cryo-EM study on PepNL reveals a distinct arrest mechanism, in which the N-terminus of PepNL folds back towards the tunnel entrance to prevent the catalytic GGQ motif of the release factor from accessing the peptidyl transferase center, causing translation arrest at the UGA stop codon. Furthermore, unlike sensory arrest peptides that require an arrest inducer, PepNL uses tryptophan as an arrest inhibitor, where Trp-tRNA^Trp reads through the stop codon. Our findings illuminate the mechanism and regulatory framework of nascent peptide-induced translation arrest, paving the way for exploring regulatory nascent peptides.

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Introduction

Translation is an essential process that converts nucleotide sequences into amino acid sequences, facilitating the expression of a diverse array of genes with various functions inside the cell. This complex process is orchestrated by the ribosome, a universal protein factory found in all cellular forms of life on Earth. In bacteria, ribosomes assemble an initiation complex upon encountering the Shine-Dalgarno sequence in mRNA upstream of the initiation codon¹. Subsequently, ribosomes catalyze the transfer of the growing peptide chain from an aminoacyl/peptidyl-tRNA at the P-site to an aminoacyl-tRNA at the A-site². Upon encountering a stop codon, peptide release factors (RF) recognize the stop codon to trigger the hydrolysis of the ester bond between the nascent peptide and tRNA^3,4,5,6,7,8. This event leads to the dissociation of the polypeptide chain and terminates translation.

While ribosomes were once considered robust protein factories capable of translating any mRNA sequence, it is now evident that this is not always the case. For example, the nascent polypeptide chain (nascent peptide) within the ribosome tunnel can cause translational difficulties^9,10,11. The nascent peptide exit tunnel (NPET) is primarily composed of negatively charged rRNA and contains a constriction site formed by ribosomal proteins¹². Certain amino acid sequences in the nascent peptide closely interact with the complex tunnel structures, causing elongation stalling, a phenomenon termed “translation arrest”^13,14. Although translation arrest appears to be disadvantageous and might be eliminated during evolution, conserved translation arrests persist among organisms, which may utilize it as part of their survival strategies^{15,16,17,18,19}.

The majority of ribosome arrest peptides (RAPs) discovered so far are encoded within upstream ORFs (uORFs)^9,10. The occurrence of translation arrest often depends on environmental changes inside and outside the cell and serves to regulate the expression of downstream genes. For example, Escherichia coli TnaC induces translation arrest only when the intracellular tryptophan concentration is high²⁰. In this arrest, excessive tryptophan enters the ribosome tunnel and alters the structure of the peptidyl transferase center (PTC) through interactions with the nascent peptide and the tunnel, thus inhibiting translation termination^21,22. In this way, TnaC regulates the expression of tryptophanase (TnaA) in a manner dependent on intracellular tryptophan as an “arrest inducer”. RAPs that control the expression of downstream genes like TnaC are widely found from prokaryotes to eukaryotes and are considered useful and universal gene expression regulation mechanisms in life^9,10,11. The search for such unidentified RAPs has been advanced through sequence and gene structure conservation analyses^23,24,25,26, as well as large-scale translatome analyses using ribosome profiling^27,28,29. However, the overall landscape of nascent peptide-dependent translation regulation remains enigmatic.

In addition to the diversity of the sequences that cause translation arrest, the molecular mechanisms of arrest also vary considerably. Recent cryo-electron microscopy (cryo-EM) structural analyses have significantly clarified the molecular details of translation arrest, revealing the intricate interactions between RAPs and the ribosome tunnel at resolutions approaching 2.0 Å^{21,22,30,31,32,33,34,35,36,37,38}. Structural studies of arrest peptides, including TnaC, SpeFL, and MsrDL have revealed case examples of their mechanisms for sensing metabolites and antibiotics, as well as their inhibition of release factors. However, structural analyses of ribosomes arrested by RAPs are currently limited to typical examples, and further studies are needed to systematically understand the mechanism of translation arrest.

In this study, we comprehensively examined whether small ORFs recently identified from ribosome profiling exhibit RAP activity. Through phenotype characterization, proteomic analysis, and mass spectrometry (MS) analyses of nascent peptides, E. coli PepNL (14 aa) and NanCL (14 aa) were demonstrated to induce translation arrest at their stop codons. Focusing on PepNL, our cryo-EM analysis revealed an unusual structure in which the nascent PepNL peptide folds back towards the entrance of the ribosome tunnel, contrary to the usual orientation. This distinctive conformation distorts the structure of the subsequent nascent peptide, leading to a steric clash with the GGQ motif of RF2 and thereby inhibiting translation termination. Unlike previously studied RAPs such as TnaC, PepNL arrested the ribosome solely based on its amino acid sequence, without requiring any arrest inducer. This study not only introduces a distinct mode of translation arrest but also provides insights into the regulatory mechanism of the PepNL-dependent arrest, which is achieved by tryptophanyl-tRNA.

Results

Screening of uncharacterized ribosome arrest peptides by overexpression phenotype

The overexpression of TnaC, a tryptophan-dependent ribosome arrest peptide (RAP), reportedly impedes cell growth due to the depletion of tRNA^Pro captured within the arrested ribosome (Fig. 1a)³⁹. To identify uncharacterized RAPs, we investigated whether the overexpression of RAPs other than TnaC would inhibit cell growth, as observed with TnaC in nutrient-rich media. Notably, the overexpression of established RAPs in E. coli cells growing on LB agar, such as SecM¹³ and TnaC¹⁴, resulted in significant growth inhibition (Fig. 1b). In contrast, SpeFL, which requires a sufficient supply of ornithine for arrest induction (Fig. 1b)³⁴, or the TnaC variant harboring the arrest-attenuating P24A mutation, failed to induce growth inhibition (Supplementary Fig. 1a). MgtL, another type of regulatory nascent peptide that triggers premature translation abortion, had a negligible effect on cell growth (Fig. 1b)⁴⁰. Based on these results, we inferred that the cytotoxicity induced upon overexpression could serve as one of the indicators of RAP activity.

In this study, we analyzed a total of 38 candidates, including 26 recently annotated small open reading frames (sORFs)^{27,28,29,41,42,43} and 12 putative sORFs identified by the GWIPS-viz browser (Supplementary Fig. 1b–d)⁴⁴. Upon overexpression of these 38 candidates in E. coli cells, 18 sORFs induced growth inhibition (Fig. 1c). The presence or absence of cytotoxicity did not correlate with their regulatory effects on downstream gene expression (Fig. 1c, colored frame, Supplementary Fig. 1e).

Subsequently, we investigated whether the examined sORFs induce stress responses due to excessive translation arrest. We anticipated that TnaC overexpression would inhibit translation elongation by tRNA^Pro depletion. In such a situation, the expression of cold shock proteins (CSPs) is expected to increase, as observed in E. coli cells exposed to sublethal concentrations of chloramphenicol (Cm) or tetracycline (Tet), which globally inhibit translational elongation^45,46,47,48. Based on this hypothesis, we conducted a comparative and quantitative proteomic analysis for E. coli cells overexpressing each of the sORFs, and analyzed their proteomic landscape rearrangement (Fig. 1d). The obtained dataset (Supplementary Data 1 and 2) was subjected to PCA (principal component analysis), and a plot illustrating the similarity of the proteomic rearrangements is depicted in Fig. 1e. Parallel clustering analysis categorized our datasets into three clusters: I. induction of CSPs expression (blue circles), II. induction of HSPs expression (red circles), and III. limited variation (green circles) (Fig. 1e, Supplementary Fig. 1f–i). Overexpression of RAPs such as TnaC and SecM, as well as treatment with translation inhibitors like Cm, induced the expression of CSPs, including CspA, DeaD, and InfC (Supplementary Fig. 1h)^49,50,51,52. These datasets were grouped in the same cluster, indicating that the inhibition of translation elongation often induces the expression of CSPs (Fig. 1eblue circles, Supplementary Fig. 1i). A similar “cold shock”-like response was observed for 12 sORF candidates, suggesting the possible occurrence of translation arrest during their expression. Meanwhile, several sORFs such as YqgB induced the expression of heat shock proteins (HSPs) that strongly respond to aggregation, including IbpA and ClpB (Fig. 1ered circles, Supplementary Fig. 1h and i)^45,53,54. Overexpression of these sORFs, unlike TnaC, might inhibit cell growth due to accumulation of the short peptides.

LC-MS/MS measurements of the peptidyl-tRNA molecules

We further evaluated the RAP activities of candidate sORFs by an LC-MS/MS analysis of the peptidyl-tRNA intermediates that accumulate due to translation arrest. After the overexpression of sORFs in E. coli cells, total RNA was extracted and purified by silica adsorption. Peptide fragments ester-bonded to tRNA were then subjected to alkaline hydrolysis, peptidase digestion, and LC-MS/MS analysis (Fig. 2a)^55,56. Identification of the MS2 spectra of the ^19thK / IVDHRP^25th fragment of TnaC (Fig. 2b) or ^149thK / GSTPVWISQAQGIRAG^165th fragment of SecM (Fig. 2c), which coincides with the established translation arrest site, demonstrates the effectiveness of this method. Additionally, the identification of Tris-adducted peptide fragments at the C-terminus, which occurs upon alkaline hydrolysis of peptidyl-tRNA, further supports the RAP activities of TnaC and SecM (Supplementary Fig. 2a and b).

**Fig. 2: Identification of the ribosome-arresting peptidyl-tRNA by LC-MS/MS.**

The LC-MS/MS analysis of peptidyl-tRNA was extended to include sORF candidates, resulting in the identification of peptide fragments indicative of stalling at various sites (Supplementary Data 3). Many of the identified peptide fragments were inconsistent with the distribution of ribosomes on the mRNA in the ribosome profiling. In contrast, the MS2 spectra indicating translation arrest at stop codons of the sORF candidates pepNL and nanCL were in line with the profiling results (Fig. 2d and e, Supplementary Fig. 1b). In addition, Tris-adducted peptide fragments at the C-terminus were detected for these two sORFs, as in the cases of TnaC and SecM (Supplementary Fig. 2c and d). Based on these LC-MS/MS results and the growth inhibition results shown in Fig. 1, we concluded that pepNL and nanCL encode RAPs that induce translation arrest at stop codons.

Structure of the PepNL-arrested ribosome

In this study, we focused on PepNL, which induced a particularly robust translation stress response (Fig. 1e). PepNL, the 14 amino acid ORF whose AUG initiation codon was verified by the reporter assay (Supplementary Fig. 3a and b), is encoded upstream of the aminopeptidase pepN gene. The frameshift mutation abolished the cytotoxicity (Fig. 3a) and the accumulation of peptidyl-tRNA (Fig. 3b), indicating that PepNL arrests translation through the context of its amino acid sequence. Moreover, the frameshift mutation increased the expression of PepN, underscoring the regulatory role of pepNL in a translation arrest-dependent manner (Supplementary Fig. 1e and 3c).

**Fig. 3: PepNL nascent peptide turns back toward the entrance of the ribosome tunnel.**

To elucidate the molecular mechanism of PepNL-dependent translation arrest, we analyzed the structure of the ribosome arrested by the PepNL nascent peptide. We translated pepNL mRNA using the reconstituted cell-free translation system (PURE system: PUREfrex v1.0)⁵⁷ deprived of tryptophan, as discussed later. A toeprint analysis confirmed the accumulation of ribosomes stalled at the stop codon of the pepNL mRNA (Fig. 3c, Supplementary Fig. 3d). This result is further supported by the alleviation of PepNL toxicity when RF2 or tRNA^Asp, both of which are trapped in the arrested ribosome, were overexpressed (Supplementary Fig. 3e). To avoid dissociation or release of the stalled ribosomes, we directly applied the in vitro translation mixture to the cryo-EM grids without purification and performed the cryo-EM data collection. The contrast of the ribosomes on the grid was sufficient for the cryo-EM analysis even without purification, resulting in the 3D reconstruction of ribosomes. We obtained four states with different components in the A and P-sites (P-tRNA only: 31%, RF2 and P-tRNA: 25%, EF-Tu•tRNA and P-tRNA: 23%, and empty: 21%, Supplementary Fig. 4). We further analyzed the particles in the RF2 and P-tRNA bound state and P-tRNA bound state, and determined their structures at 2.9 Å resolution (Fig. 3d, Supplementary Fig. 5). In the structure determined with RF2, the arrested 70S ribosome was bound with the mRNA, RF2 at the A-site, and tRNA^Asp carrying the nascent PepNL peptide at the P-site (Supplementary Fig. 6a).

All 14 amino acid residues of PepNL were successfully traced in the density, indicating that the PepNL nascent peptide adopts a stable conformation in the exit tunnel. The PepNL peptide forms a mini-hairpin conformation with residues Lys2 to Ala10, which directs the N-terminal residues back toward the PTC but not far enough to reach the constriction site of the exit tunnel (Fig. 3e and f). This distinctive structure is stabilized by intramolecular interactions, including a hydrophobic interaction between Ile3 and Tyr9 (Fig. 3g, panel 1), as well as β-sheet-like main-chain interactions involving Lys2 with Ala10 and Leu4 with Ile8 (Fig. 3g, panel 2). Moreover, intermolecular hydrophobic interactions between PepNL (Ile3, Leu4, Ile8, and Tyr9) and 23S rRNA (U2609, A2062, A2058, A2059, and U2610) also contribute to supporting the structure (Fig. 3h). While the N-terminal portion of PepNL exhibits a compact conformation, the C-terminal part is distorted, as illustrated by a large main-chain shift as compared to nascent peptides that lack RAP activity⁵⁸ (Fig. 3i) {PDB: 8CVJ (nascent peptide sequence: fMSEAC) and 8CVL (fMTHSMRC)}. The distortion of PepNL further interrupts the hydrogen-bond network between rRNA (A2506 and A2062) and the nascent peptides without RAP activity, resulting in the conformational rearrangement of A2506 and A2062 in the 23S rRNA (Supplementary Fig. 6b and c). These structural observations are supported by the fact that the arrest activity of PepNL was abolished by alanine substitutions of Ile3, Leu4, Ile8, and Tyr9, which are involved in the formation of the β-hairpin loop (Fig. 3j, Supplementary Fig. 3f).

RF2 undergoes a conformational rearrangement

We next focused on the structural rearrangement of RF2. In canonical translation termination, the recognition of the stop codon by domain II of RF2 triggers the positional extension of domain III, inserting the ^250thGGQ^252nd catalytic motif into the PTC to hydrolyze the ester bond of the peptidyl-tRNA (Fig. 4a)^5,6,7,8. Within the PepNL-arrested ribosome, domain II of RF2 properly recognizes the UGA stop codon and the extension of domain III is also observed (Fig. 4a). However, we found a significant difference in the conformation of the apical loop (residues 246-257) in a comparison of our structure with the canonical termination complex⁵⁹ (PDB: 6C5L) (Fig. 4b). In the canonical structure, the methylated Gln252 in the GGQ motif enters the narrow pocket formed by A2451, C2452, and U2506 of rRNA (Figs. 4b and f). However, in the arrested structure, Gln252 undergoes a 16 Å relocation, as measured between the Nε2 atoms (Fig. 4b). This relocation causes a drastic rearrangement of the apical loop, which adopts an inactive conformation stabilized by hydrogen bonds between the residues in the apical loop (Gly251, Gly252, and Arg256) and rRNA nucleotides (Fig. 4c). In addition to the conformational change of the apical loop, we observed a notable shift of the entire domain III of RF2, by approximately 3 Å toward the L1 stalk, in comparison with its position in the canonical termination complex (Figs. 4a and b). Residues within the shifted region (Arg245, Glu258, Arg262, Gln280, and His281) further stabilize this conformation through hydrogen-bonding interactions with rRNA nucleotides (Figs. 4c–e). Since there are several hydrogen-bonding interactions between domain III and rRNA that are absent in the canonical termination complex, the rearranged and shifted RF2 conformation observed in our structure could be functionally important for general termination regulation.

**Fig. 4: PepNL blocks RF2-mediated translation termination.**

The PepNL nascent peptide inhibits RF2 activity through a steric clash with Ile13

Further structural comparisons showed that the side chain of Ile13 in the distorted C-terminus of PepNL nascent peptide is accommodated in the pocket formed by A2451, C2452, and U2506 (Fig. 4f). This pocket is crucial for translation termination, as the ^250thGGQ^252nd motif of RF2 enters this pocket to cleave the ester bond^5,6,7,8,59. Consequently, the distortion of Ile13 shown in Fig. 3i leads to a steric clash with Gln252 of RF2, hampering the accommodation of the GGQ motif in the A2451/C2452/U2506 pocket (Fig. 4f). This steric clash between Ile13 of PepNL and Gln252 of RF2 would induce a drastic rearrangement of the apical loop and the subsequent rearrangement of the entire domain III of RF2. These considerations are supported by the finding that the Ile13 mutation abolished the arrest activity of PepNL (Fig. 3j, Supplementary Fig. 3f).

Taken together, our cryo-EM reconstruction revealed the molecular details underlying the PepNL nascent peptide-mediated inhibition of the translation termination activity of RF2. The translated nascent PepNL peptide forms a compact hairpin structure, with its N-terminus directed back toward the PTC, and stacks inside the exit tunnel (Fig. 4g, left). This stacking distorts the C-terminus of the PepNL nascent peptide, leading to the steric clash between Ile13 of PepNL and Gln252 of RF2 (Fig. 4g, middle). Consequently, this steric clash induces a drastic conformational change in RF2, shifting it into an inactive conformation (Fig. 4g, right).

Read-through of the stop codon serves as an inhibitory mechanism for PepNL-induced translation arrest

Despite our success in determining the structure of the PepNL-arrested ribosome, we initially faced difficulties in preparing the stalled ribosome within the complete PUREfrex system (Fig. 5a, lanes 1 and 2). However, our experiments with the pepNL mutant, harboring a UAG stop codon instead of UGA, revealed robust ribosome stalling both in vitro and in vivo (Fig. 5a, lanes 5 and 6, Supplementary Fig. 7a, and b). Furthermore, the pepNL mutants carrying the altered stop codon exhibited increased cytotoxicity (Supplementary Fig. 7c). These results indicated that the UGA stop codon of pepNL plays a role in attenuating ribosome stalling.

**Fig. 5: Read-through of stop codon serves as an arrest inhibition mechanism for PepNL.**

To investigate this, we conducted experiments where Trp-tRNA^Trp, which is known to induce read-through at the UGA codon^60,61,62, was depleted from the in vitro translation system by excluding tryptophan. Remarkably, even with the wild-type pepNL (UGA), robust ribosome stalling was observed (Fig. 5a, lanes 3 and 4). We further analyzed the polypeptide products and detected an accumulation of the peptidyl-tRNA with a 14-aa-length polypeptide when tryptophan was absent, consistent with the toeprint analysis. (Fig. 5b and c, lanes 3, 4, 5 and 6). Contrary, in the presence of tryptophan, we observed a longer peptidyl-tRNA compared to full-length PepNL (14 aa)-tRNA (Fig. 5c, lane 1). This longer peptidyl-tRNA exhibited almost identical electrophoretic mobility to the peptidyl-tRNA containing 27-aa-length polypeptide up to the downstream stop codon (Fig. 5c, lane 7, Supplementary Fig. 7a). These results indicated that ribosomes could read through the UGA stop codon in the presence of the Trp-tRNA^Trp and then stall at downstream stop codons. Consistent with this, in vivo reporter assays demonstrated that the UGA codon, but not UAA or UAG, exclusively induced the read-through of the pepNL stop codon (Fig. 5d). Moreover, when we mutated the UGA15 stop codon of pepNL to those of 20 canonical amino acids, we observed varying degrees of inhibition in the peptidyl transfer of the 14-amino-acid-long PepNL nascent peptide. Mutations to Asp and Glu resulted in pronounced inhibition, while Ile, Lys, Leu, Asn, Pro, Arg, Val, and Trp showed moderate inhibitory effects. The remaining amino acid mutations exhibited little or no inhibition (Supplementary Fig. 7d). A similar observation, where peptidyl transfer inhibition depends on the identity of the A-site aminoacyl-tRNA, has been reported previously^63,64. Nonetheless, these data indicate that PepNL nascent peptide blocks not only RF2-mediated termination but also interferes with transpeptidation involving specific aminoacyl-tRNAs at the A-site.

Finally, the addition of tryptophan triggered stop codon read-through even after the ribosome was arrested by PepNL (Fig. 5e). However, the migration rate under these conditions is relatively slow (approximately one aa/min). Moreover, toeprint analysis revealed that the read-through becomes less efficient when tryptophan is reintroduced after depletion, with ribosome progression halted at multiple sites beyond the UGA15 stop codon (Supplementary Fig. 7e). Structural comparison further revealed that Ile13 in the hairpin-shaped PepNL potentially clashes with not only RF2 but also Trp-tRNA^Trp (Supplementary Fig. 7f). Collectively, these findings suggest that Trp-tRNA^Trp reads through the UGA15 stop codon more efficiently “before” the formation of the PepNL’s mini hairpin (Fig. 5f, discussed in the following section).

Discussion

Our study demonstrated that the small ORFs pepNL and nanCL in E. coli encode ribosome arrest peptides (RAP) (Figs. 1 and 2). Furthermore, we successfully determined the cryo-EM structure of the ribosome arrested by PepNL (Figs. 3 and 4). Our structure revealed that the nascent PepNL peptide adopts a distinctive “mini hairpin” conformation near the entrance of the exit tunnel, causing the distortion of its C-terminal segment, including the Ile13 residue. This distorted structure prevents the proper accommodation of the GGQ motif of RF2, inhibiting peptide release. Importantly, PepNL-dependent stalling does not require an “arrest inducer”; instead, the stalling is inhibited via stop codon read-through induced by Trp-tRNA^Trp (Fig. 5).

The PepNL nascent peptide within the tunnel adopts a mini-hairpin fold to enable a distinct arresting mechanism, by employing only 14 amino acid residues. Generally, the N-terminus of the nascent peptide elongates toward the exit of the tunnel^9,12,58. In contrast, in our structure, the N-terminus of PepNL is oriented in the opposite direction, toward the tunnel entrance. In the previous studies, the nascent peptides of VemP, XBP1u, and MsrDL were reported to adopt S-shaped or hook-like conformations that partially folded back within the exit tunnel^32,33,36. However, a U-shaped conformation with the overall reversal of the nascent peptide, as in the present PepNL structure, has not yet been reported. Moreover, the translation inhibition mechanism by this “mini hairpin” structure might raise a concept at the early stage of protein synthesis. The N-terminal amino acid sequences immediately adjacent to the initiation codon can affect translation efficiency^65,66. Therefore, the stochastic formation of mini hairpin-like structures could potentially inhibit the expression of proteins, and thus not be limited to PepNL.

Previous cryo-EM studies elucidated the details of arrest peptide prevention of translation termination^21,22,33,36. In the case of TnaC, U2585 and A2602 of the 23S rRNA and Arg23 (Phe23) of the TnaC nascent peptide cooperatively inhibit the proper accommodation of RF2²². In SpeFL, the distortion of U2585 by Asn32 of the SpeFL nascent peptide causes a steric clash with RF1, thereby inhibiting peptide release³³. In MsrDL, U2585 and U2584 of the 23S rRNA and Phe5 of the MsrDL nascent peptide prevent the accommodation of RF1 and RF2³⁶. In the present structure, the steric clash between Ile13 of the PepNL nascent peptide and the GGQ motif of RF2 inhibits peptide release. These four RAPs commonly prevent the proper accommodation of the GGQ motif by forming distinct compact conformations (Supplementary Fig. 8a–c). Moreover, our study once again emphasizes the significance of the penultimate amino acid residue (Ile13 in PepNL) for peptide release inhibition^22,67. The structural comparison of PepNL with other RAPs that inhibit translation termination revealed that, although the overall shape of the nascent peptides is distinct from one another, the distortion of the penultimate amino acid shows a striking similarity (Supplementary Fig. 8d–f). Earlier studies noted that the efficiency of RF-mediated termination is affected by the amino acid sequence of the nascent peptide^68,69,70. Notably, Isaksson and colleagues pointed out the importance of the penultimate C-terminal residue in canonical translation termination⁶⁷. The inhibition mechanisms revealed by recent structural analyses may contribute to a deeper understanding of the universal translation cycle. Our successful examination of the entire structure of distorted RF2 could offer several insights. We observed that Ile13 of PepNL excluded the GGQ motif, causing a drastic rearrangement of the apical loop within domain III of RF2. In addition, this noncanonical structure of RF2 is stabilized by extra hydrogen bonds (Fig. 4c–e). Therefore, the GGQ motif can be regarded as a sensor to fine-tune the translation termination efficiency in response to the structural state within the PTC. When a mismatch exists between the GGQ motif and the PTC structure, it could result in the stabilization of the non-canonical RF2 conformation via additional interactions, potentially delaying translation termination.

In contrast to sensory arrest peptides such as TnaC, the folding of the PepNL nascent peptide within the tunnel is independent of an arrest inducer. Instead, PepNL utilizes the stop codon read-through as an “arrest inhibitor”. This framework makes PepNL a metabolite-sensing arrest peptide that functions in a distinct way from the previously studied RAPs. However, the PepNL nascent peptide, which forms a mini hairpin, could cause steric clashes not only with RF2 but also with Trp-tRNA^Trp (Supplementary Fig. 7f). Furthermore, after stop codon read-through, the ribosome exhibited a slower migration rate (approximately one aa/min, Fig. 5e) compared to the reported elongation velocity in the PURE system (4.8 ~ 26.4 aa/min^71,72,73). These suggest that the hairpin structure may inhibit not only the termination process but also stop codon read-through and subsequent translation elongation (Fig. 5e, Supplementary Fig. 7e). These together indicate that the read-through hardly releases the PepNL-arrested ribosome. What accounts for this discrepancy? Although we have not obtained direct evidence in this study, we predict that the PepNL nascent peptide does not form the hairpin structure immediately after synthesis (the pre-arrest state in Fig. 5f). At this stage, Trp-tRNA^Trp could efficiently read through the UGA15 stop codon, thereby preventing translation arrest (Fig. 5f, top). However, once the hairpin structure forms, it inhibits termination by RF2 and read-through by Trp-tRNA^Trp (Fig. 5f, bottom). Therefore, since PepNL relies on its ~~unique~~ hairpin structure for translation arrest, PepNL may utilize the “time” required for folding as an arrest inducer. This concept is reminiscent of how SpeFL relies on non-optimal rare codons for effective regulation, potentially promoting the folding of its N-terminal sensor domain by slowing translation³⁴. Further analyses are necessary to clarify this hypothesis in greater detail.

The translation of pepNL regulates the expression of the downstream gene, pepN (Supplementary Fig. 1e, 3c). The ViennaRNA prediction program^74,75 indicated the presence of stem-loop structures surrounding the stop codon of pepNL, as well as near the Shine-Dalgarno (SD) sequence and the initiation codon of pepN (Supplementary Fig. 9a). However, the SD-masking stem-loop does not seem to be affected by translation arrest at the pepNL stop codon (Supplementary Fig. 9b). Further studies are needed to clarify the detailed regulatory mechanism involved. PepN, an endopeptidase, degrades not only oligopeptides but also full-length polypeptides with broad substrate specificity⁷⁶. In nutrient-limited conditions, PepN plays a crucial role in adaptation through the reproduction of free amino acids⁷⁷. These characteristics suggest that the correlation between the PepN activity and the intracellular amino acid concentration holds significance for environmental adaptation.

Our approaches identified two uncharacterized RAPs, PepNL and NanCL, with functions in living E. coli cells. However, it should be noted that our evaluations of the RAP activity for each sORF are limited to the standard laboratory conditions (LB medium, 37 °C, aerobic conditions). In fact, SpeFL, which responds to ornithine, did not exhibit a significant result under our testing conditions, indicating the limitations of our approaches. Meanwhile, our analysis did not exclude the possibility that sORFs other than pepNL and nanCL encode sensory arrest peptides. In our study, the translation of 19 out of 39 sORFs significantly influenced the expression of downstream genes, implying that they harbor translation-coupled functions (Supplementary Fig. 1e). Further analyses of these sORFs would expand our understanding of translation regulation.

To explore the unidentified “arrest inducer” for each RAP candidate, monitoring the expression of Cold Shock Proteins (CSPs) could serve as a valuable indicator. While the stalling of translation elongation commonly triggers the expression of CSPs, the precise mechanism has not yet been elucidated⁴⁵. This enigmatic phenomenon should be studied in the future, and if similar responses occur across species^78,79, then it could be a convenient method for evaluating RAP activity. In addition, this study also demonstrated the effectiveness of LC-MS/MS measurements of nascent peptidyl-tRNA species to identify RAPs. Our approaches to identifying PepNL and NanCL, as well as the distinct molecular mechanism of translation stalling and regulation, provide valuable insights into deciphering the hidden genetic codes within polypeptide sequences.

Methods

E. coli strains, plasmids, and primers

E. coli strain BW25113 {∆(araD-araB)567, ∆lacZ4787(::rrnB-3), λ-, rph-1, ∆(rhaD-rhaB)568, hsdR514} was used as the experimental standard strain. Plasmids used in this study are listed in Supplementary Data 4. Plasmids were constructed using standard cloning procedures, including Gibson assembly and site-directed mutagenesis. The sequence files of the plasmids constructed in this study are available in the Mendeley repository [https://data.mendeley.com/datasets/2bkz2xnnn5/1].

Spot assay

E. coli cells harboring pCA24N with each sORF were grown overnight at 37 °C in LB medium supplemented with 20 µg/ml chloramphenicol. On the next day, cultures were serially diluted with fresh LB medium (10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶) and spotted onto LB agar plates containing 20 µg/ml chloramphenicol, with or without 1 mM IPTG (isopropyl β-D-thiogalactopyranoside, Nacalai Tesque). The plates were incubated overnight at 37 °C, and the growth of colonies was recorded.

In the complementation assay shown in Supplementary Fig. 3e, E. coli cells harboring pCA24N-pepNL and pBAD30 or its derivatives harboring the indicated genes under the control of the P_BAD promoter were grown overnight at 37 °C in LB medium supplemented with 20 µg/ml chloramphenicol and 100 µg/ml ampicillin. On the next day, cultures were serially diluted and spotted onto LB agar plates containing 20 µg/ml chloramphenicol, 100 µg/ml ampicillin, and 2×10⁻³ % arabinose, with or without 500 µM IPTG. Plates were incubated overnight at 37 °C, and the growth of colonies was recorded.

β-galactosidase assay

E. coli cells harboring the lacZ reporter plasmid were grown overnight at 37 °C in LB medium supplemented with 100 µg/ml ampicillin. On the next day, the cultures were inoculated into fresh LB medium containing 2 ×10⁻³% arabinose and 100 µg/ml ampicillin, and grown at 37 °C until the A₆₆₀ reached 0.5. Afterward, 20 µl portions were subjected to a β-galactosidase assay to calculate the Miller units (m.u.), as described⁸⁰. The average of three independent experiments is presented with a standard error (SE) value.

sORF-dependent regulation score

The sORF-dependent regulation score was calculated using the following formula.

$$ {\rm{sORF}}-{{\rm{dependent}}\; {\rm{regulation}}\; {\rm{score}}}\\ =\{{\rm{m.u.}}\,({\rm{Plasmid}}-{\rm{B}})\}/[\{{\rm{m.u.}}\,({\rm{Plasmid}}-{\rm{B}})\}+{\rm{m.u.}}\,({\rm{Plasmid}}-{\rm{A}})\left\}\right.]$$

Plasmid-A: A plasmid carrying the P_BAD promoter, the 5’ region of the downstream main ORF (mORF) relative to the small ORF, spanning from the transcription start site to the initiation codon of the mORF, and the lacZ reporter translationally fused with the mORF. Plasmid-B: A derivative of plasmid-A with a mutation (ATG to ACG) to disrupt the translation initiation codon (AUG) or the insertion of a stop codon to prematurely terminate the translation of the small ORF. Detailed information on these mutations can be found in Supplementary Data 4, and sequence files are available in the Mendeley repository [https://data.mendeley.com/datasets/2bkz2xnnn5/1].

Frequency of stop codon read-through

E. coli cells expressing the pepNL-stop-lacZ or its derivative lacking the stop codon of the pepNL reporter were grown and subjected to a β-galactosidase assay. The frequency of stop codon read-through was calculated with the following formula.

$$\begin{array}{c}{\bf{Stop}}\,{\bf{condon}}\,{\bf{read}}-{\bf{through}}\, ({\boldsymbol{\%}})\\={\rm{m}}.{\rm{u}}.\,(pepNL-{\rm{stop}}-lacZ)/{\rm{m}}{\rm{.u}}.\,(pepNL\varDelta stop-lacZ)\end{array}$$

In vitro translation and product analysis

The coupled transcription-translation reaction was performed using PUREfrex v1.0 (GeneFrontier) in the presence of ³⁵S-methionine {EasyTag L-[³⁵S]-Methionine (PerkinElmer)} at 37 °C, as described previously⁴⁰. DNA templates were prepared by PCR, as summarized in Supplementary Data 5. Tryptophan or release factors (RF: RF1, RF2, RF3, and RRF) were excluded from the in vitro translation mixture where indicated. The reaction was stopped by the addition of ice-cooled 5% TCA, washed with ice-cooled acetone, and dissolved in SDS sample buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT) that had been treated with RNAsecure (Ambion). Finally, the sample was divided into two portions, one of which was incubated with 50 µg/ml of RNase A (Promega) at 37 °C for 60 min, and separated by a WIDE RANGE Gel SDS-PAGE system (Nacalai Tesque). Radioactive bands were detected by using an imaging plate and an Amersham™ Typhoon™ scanner RGB system (GE Healthcare).

Toeprint analysis

The toeprint analysis was performed as described previously^40,81. The in vitro translation reaction sample was mixed with an equal volume of reverse transcription mixture [50 mM HEPES-KOH, pH 7.6, 100 mM potassium glutamate, 2 mM spermidine, 13 mM magnesium acetate, 1 mM DTT, 2 µM fluorescently labeled oligonucleotide (pe-lacZ-N-rv with Alexa 647 at 5’-terminus), 40 µM of each dNTP, 10 unit/µl ReverTra Ace (Toyobo)] and incubated at 37 °C for 10 min. The reverse transcription products were purified by a NucleoSpin Gel and PCR clean-up kit equilibrated with NTC buffer (Macherey-Nagel). Dideoxy DNA sequencing samples were prepared using Thermo Sequenase DNA polymerase (Cytiva) and the same templates and primer (pe-lacZ-N-rv) as used for the toeprint analysis. Samples were subjected to 8% polyacrylamide-7 M urea-TBE gel electrophoresis. Fluorescent images were visualized and analyzed by an Amersham™ Typhoon™ scanner RGB system (GE Healthcare), using a 635 nm excitation laser and LPR emission filter. Thiostrepton was added at a final concentration of 100 µg/ml beforehand, as indicated. Tryptophan or release factors (RFs: RF1, RF2, RF3, and RRF) were excluded from the in vitro translation mixture as indicated.

Northern blotting

E. coli cells were grown in LB medium until the A₆₆₀ reached ~0.5. The expression of pepNL was then induced by 1 mM IPTG for 10 min. After centrifuging and collecting the bacterial cells, total RNA was extracted using TriPure Isolation Reagent (Roche), according to the supplier’s instructions. RNA samples were dissolved in SDS sample buffer, separated by 11% WIDE RANGE Gel SDS-PAGE, transferred onto a BrightStar™-Plus Positively Charged Nylon Membrane (Invitrogen^TM), and hybridized with a biotinylated oligonucleotide complementary to tRNA^Asp; CGGAACGGACGGGACTCGAACCCGCGACC. Hybridization experiments were performed using ULTRAhyb™ Ultrasensitive Hybridization Buffer (Invitrogen^TM) and the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific), according to the manufacturers’ instructions. Images were visualized and analyzed by a LAS4000 LuminoImager (GE Healthcare).

Preparation of the lysate for proteomic analysis

Samples for proteomic analysis were prepared as described previously⁸². E. coli cells harboring pCA24N with each sORF were grown in LB medium supplemented with 20 µg/ml of chloramphenicol until the A₆₆₀ reached ~0.2. Subsequently, 1 mM of IPTG was added to induce the expression of small ORFs, and cells were further incubated for 1 h with shaking at 37 °C. The cells were then harvested and resuspended in PBS buffer (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄, pH 7.4). The suspension was mixed with an equal volume of 10% TCA. After standing on ice for at least 10 min, the samples were centrifuged, and the supernatant was removed by aspiration. Precipitates were washed twice with acetone, by vigorous mixing. Proteins were dissolved in PTS buffer (12 mM sodium deoxycholate, 12 mM sodium lauryl sulfate, 100 mM Tris-HCl, pH 9.0), and 50 µg of total protein at a concentration of 1 µg/µl was processed by reduction with dithiothreitol (DTT), alkylation with iodoacetamide, and limited digestion by Trypsin/Lys-C Mix (Promega). The samples were then extracted with ethyl acetate, evaporated, dissolved in MS buffer-A (0.1 % TFA and 2% acetonitrile), desalted by a StageTip composed of an SDB-XC Empore disk (3 M, U.S.A.), eluted with MS buffer-B (0.1% TFA and 80% acetonitrile), evaporated again, and dissolved in MS buffer-A. Finally, the samples were subjected to the LC-MS/MS measurement.

LC-MS/MS analysis for comparative quantitative proteomic analysis

In a series of quantitative proteomic analyses, we used the Eksigent NanoLC 415 nanoflow HPLC system and the TripleTOF 4600 tandem-mass spectrometer (AB Sciex, U. S. A.) in the DIA/SWATH acquisition mode⁸³. The detailed settings for the LC-MS/MS measurements are summarized in Supplementary Table 1. The measurement was conducted three times for each sample prepared from one biological replicate.

Data analysis was performed by the DIA-NN software (version 1.8.1, https://github.com/vdemichev/diann, downloaded on 1 November 2023)⁸⁴. The spectral library for DIA/SWATH analysis was obtained from the SWATH atlas (http://www.swathatlas.org/, accessed on 30 April 2021); the original data were acquired by Midha et al.⁸⁵. All downstream statistical analyses were performed using in-house R scripts (R.app for Mac, version 4.3.1). Only the proteins with intensities obtained in all three measurements in both samples were used to calculate fold changes. P-values for the volcano plots were calculated using Welch’s t-test with the Benjamini-Hochberg correction (using the “p.adjust” function in R.app). PCA and hierarchical clustering analyses were performed using the data from 1,205 proteins for which foldchange values were obtained under all 36 conditions.

Preparation of the peptidyl-tRNA for LC-MS/MS analyses

E. coli cells harboring pCA24N with each sORF were grown in LB medium supplemented with 20 µg/ml of chloramphenicol until the A₆₆₀ reached approximately 0.5. Subsequently, 1 mM of IPTG was added to induce the expression of small ORFs, and cells were further incubated for 30 min with shaking at 37 °C. Afterward, the cell culture was mixed with an equal volume of ice-cooled 10% TCA to precipitate the cellular components. The precipitate was washed twice with ice-cooled acetone, and dissolved in PTS buffer.

The peptidyl-tRNAs in the PTS buffer were isolated by using a High Pure miRNA Isolation Kit (Roche), as described previously⁵⁶. After adding binding buffer (provided by the manufacturer), the lysate was vortexed and incubated at 37 °C for 30 min with shaking. Then binding enhancer (provided by the manufacturer) was added and the lysate was centrifuged at 12,000 × g for 3 min at 4 °C to remove the precipitates. The supernatant was loaded into silica columns, and the following steps were performed according to the manufacturer’s instructions. The silica-bound RNA was eluted with PTS buffer. This purification was repeated twice.

The purified RNA sample was mixed with 0.1x volume of 2 M Tris base (pH= ~11) and incubated at 80 °C for 20 min to hydrolyze the ester bond of the peptidyl-tRNA. After the addition of 0.9x volume of deionized water, the sample was reduced by DTT, alkylated with iodoacetamide, and digested with Lys-C (Fujifilm-Wako) or Lys-C / Trypsin mix (Promega). The samples were then extracted with ethyl acetate, dried, dissolved in MS buffer-A (0.1% TFA and 2% acetonitrile), desalted by a StageTip composed of an SDB-XC Empore disk, eluted with MS buffer-B (0.1% TFA and 80% acetonitrile), dried again, and resolved in MS buffer-A.

LC-MS/MS analysis for peptides derived from peptidyl-tRNAs

For the detection of the peptides derived from peptidyl-tRNAs, the Easy-nLC 1000 nanoflow HPLC system and the Q-Exactive tandem-mass spectrometer (Thermo Fisher Scientific, U. S. A.) were used in the data-dependent acquisition (DDA) mode. The detailed settings for the LC-MS/MS measurements are summarized in Supplementary Table 2. The data analysis of the DDA measurements was performed with the Proteome Discoverer 2.4 software bundled with the SEQUEST HT search engine (Thermo Fisher Scientific, U. S. A.). To detect stalled peptides in translation, the Trypsin (semi) or LysC (semi) setting was used for enzymatic digestion.

Statistical analyses

Statistical analyses were conducted by using R and Python (https://www.python.org).

GWIPS-viz browser

We configured the settings of the GWIPS-viz browser⁴⁴ as follows: Organism: Escherichia coli K12; Elongating Ribosome (A-site): Global Aggregates; mRNA-seq Reads: Global Aggregates.

Grid preparation and data acquisition

For the cryo-EM analysis, 3 µL of the PURE system reaction solution containing stalled ribosomes was applied onto a glow-discharged holey-carbon grid coated with a continuous 2 nm-thick carbon film (Quantifoil Au 300 mesh, R2/1 + 2 nm C) and incubated for 30 s in a controlled environment of 100% humidity at 4°C. The grids were blotted for 4 s, and then plunge frozen in liquid ethane, using a Vitrobot Mark IV (FEI).

The datasets were collected using a Titan Krios G4 microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF). A Gatan K3 Summit direct electron detector was used at a pixel size of 0.83 Å (magnification of × 105,000) with a dose of approximately 30 electrons per Å ² with 30 frames. The data were automatically acquired using the EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −1.6 µm, and 15,342 movies were obtained. Detailed parameters are listed in Supplementary Table 3.

Image processing

The data were processed by the cryoSPARC v4.4.0 software platform⁸⁶. The dose-fractionated movies were subjected to beam-induced motion correction and dose weighting using patch motion correction, and the contrast transfer function (CTF) parameters were estimated using patch-based CTF estimation. From the 15,342 preprocessed micrographs, 2,521,444 particles were automatically picked by the template picker, using the references created by the blob picker and several rounds of 2D classification. The particles were subjected to several rounds of reference-free 2D classifications to create particle sets. The curated particles were aligned by non-uniform refinement using the E. coli ribosome map (EMD-22586) as the reference, followed by unsupervised 3D classification and a successive 2D classification to eliminate the ratcheted ribosomes and junk particles. The remaining particles were further classified by no-align focused 3D classification using the mask covering the P-site, A-site, and the binding sites of EF-Tu:tRNA and RF2, resulting in four classes with RF2-bound, A-tRNA-bound, EF-Tu:tRNA-bound, and empty ribosomes, respectively. The particles of the RF2-bound ribosome (71,980 particles) were subjected to CTF refinement (beam-tilt, trefoil, fourth-order aberrations, and per-particle defocus) and reference-based motion correction, followed by the final non-uniform refinement. The global resolution of the final map is 2.9 Å based on the gold-standard, applying the 0.143 criterion on the Fourier shell correlation (FSC) between the reconstructed half maps⁸⁷.

Model building and refinement

The reported high-resolution model of the E. coli ribosome⁸⁸ (PDB 7K00), including bound ligands and metal ions, was used as the starting model, while that of RF2 was from 6OUO. The models were docked into our map by rigid body fitting, followed by jiggle fitting⁸⁹ and manual revision in Coot⁹⁰. The PepNL peptide was built manually based on the density. Metal ions were revised and added based on the coordination pattern and the density, which is stronger in the map without sharpening due to their positive charge. Geometrical restraints of modified residues and ligands were calculated by Grade Web Server (http://grade.globalphasing.org). The final model was subjected to the refinement of energy minimization and atomic displacement parameters (ADP) estimation by Phenix.real_space_refine v1.20⁹¹ with Ramachandran and rotamer restraints, against the map with B-factor sharpening and local-resolution filtering by cryoSPARC. Metal-coordination restraints were generated by ReadySet implemented in PHENIX, and non-canonical covalent bond restraints for modified residues were manually prepared. The refined model was validated with MolProbity⁹² and EMRinger⁹³ in the PHENIX package. The models used for the structure comparison are docked into our map by rigid body fitting. Model refinement statistics are listed in Supplementary Table 3. UCSF ChimeraX 1.6⁹⁴ was used to make the figures.

RNA secondary structure prediction in silico

The secondary structure of pepNL-pepN mRNA was predicted using the Vienna RNAfold WebServer^74,75 with the following settings: Folding Constraints: no constraint; Fold algorithms and basic options: minimum free energy and partition function; Dangling end options: dangling energies on both sides of a helix in any case; Energy Parameters: RNA parameters (Turner model, 2004); Temperature: 37 °C; Salt concentration: 1.021 M NaCl; Modified Bases: no modification; SHAPE reactivity data: no designation.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The mass spectrometry data have been deposited in the jPOST repository with the dataset identifier PXD052430 [https://repository.jpostdb.org/entry/JPST003118.1]⁹⁵. The atomic coordinates have been deposited in the Protein Data Bank under the accession codes 8ZTU and 8ZTV. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes of EMD-60473 and EMD-60474. Source Data are provided with this paper. Raw data files are available in the Mendeley Data repository [https://data.mendeley.com/datasets/2bkz2xnnn5/1]. Source data are provided with this paper.

References

Shine, J. & Dalgarno, L. Determinant of cistron specificity in bacterial ribosomes. Nature 254, 34–38 (1975).
Article ADS CAS PubMed MATH Google Scholar
Rodnina, M. V. Translation in Prokaryotes. CSH Perspect. Biol. 10, a032664 (2018).
Google Scholar
Capecchi, M. R. Polypeptide chain termination in vitro: isolation of a release factor. Proc. Natl. Acad. Sci. Usa. 58, 1144–1151 (1967).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Scolnick, E., Tompkins, R., Caskey, T. & Nirenberg, M. Release factors differing in specificity for terminator codons. Proc. Natl. Acad. Sci. Usa. 61, 768–774 (1968).
Article ADS CAS PubMed PubMed Central Google Scholar
Weixlbaumer, A. et al. Insights into translational termination from the structure of RF2 bound to the ribosome. Science 322, 953–956 (2008).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Laurberg, M. et al. Structural basis for translation termination on the 70S ribosome. Nature 454, 852–857 (2008).
Article ADS CAS PubMed MATH Google Scholar
Korostelev, A. et al. Crystal structure of a translation termination complex formed with release factor RF2. Proc. Natl. Acad. Sci. Usa. 105, 19684–19689 (2008).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Korostelev, A., Zhu, J., Asahara, H. & Noller, H. F. Recognition of the amber UAG stop codon by release factor RF1. EMBO J. 29, 2577–2585 (2010).
Article CAS PubMed PubMed Central Google Scholar
Ito, K. & Chiba, S. Arrest Peptides: Cis-Acting Modulators of Translation. Annu. Rev. Biochem. 82, 171–202 (2013).
Article CAS PubMed MATH Google Scholar
Dever, T. E., Ivanov, I. P. & Sachs, M. S. Conserved Upstream Open Reading Frame Nascent Peptides that Control Translation. Annu. Rev. Genet. 54, 1–28 (2020).
Article Google Scholar
Chiba, S., Fujiwara, K., Chadani, Y. & Taguchi, H. Nascent chain-mediated translation regulation in bacteria: translation arrest and intrinsic ribosome destabilization. J. Biochem. 173, 227–236 (2023).
Article CAS PubMed Google Scholar
Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. The Structural Basis of Ribosome Activity in Peptide Bond Synthesis. Science 289, 920–930 (2000).
Article ADS CAS PubMed Google Scholar
Nakatogawa, H. & Ito, K. The Ribosomal Exit Tunnel Functions as a Discriminating Gate. Cell 108, 629–636 (2002).
Article CAS PubMed MATH Google Scholar
Gong, F. & Yanofsky, C. Instruction of Translating Ribosome by Nascent Peptide. Science 297, 1864–1867 (2002).
Article ADS CAS PubMed MATH Google Scholar
Gaba, A., Wang, Z., Krishnamoorthy, T., Hinnebusch, A. G. & Sachs, M. S. Physical evidence for distinct mechanisms of translational control by upstream open reading frames. EMBO J. 20, 6453–6463 (2001).
Article CAS PubMed PubMed Central Google Scholar
Onouchi, H. et al. Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis. Genes Dev. 19, 1799–1810 (2005).
Article CAS PubMed PubMed Central MATH Google Scholar
Chiba, S., Lamsa, A. & Pogliano, K. A ribosome-nascent chain sensor of membrane protein biogenesis in Bacillus subtilis. EMBO J. 28, 3461–3475 (2009).
Article CAS PubMed PubMed Central Google Scholar
Yanagitani, K., Kimata, Y., Kadokura, H. & Kohno, K. Translational Pausing Ensures Membrane Targeting and Cytoplasmic Splicing of XBP1u mRNA. Science 331, 586–589 (2011).
Article ADS CAS PubMed Google Scholar
Ishii, E. et al. Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria. Proc. Natl. Acad. Sci. Usa. 112, E5513–E5522 (2015).
Article CAS PubMed PubMed Central MATH Google Scholar
Cruz-Vera, L. R., Rajagopal, S., Squires, C. & Yanofsky, C. Features of Ribosome-Peptidyl-tRNA Interactions Essential for Tryptophan Induction of tna Operon Expression. Mol. Cell 19, 333–343 (2005).
Article CAS PubMed Google Scholar
Su, T. et al. Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide. Nucleic Acids Res. 49, 9539–9547 (2021).
Article CAS PubMed PubMed Central Google Scholar
van der Stel, A.-X. et al. Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling. Nat. Commun. 12, 5340 (2021).
Article ADS PubMed PubMed Central MATH Google Scholar
Takahashi, H. et al. Comprehensive genome-wide identification of angiosperm upstream ORFs with peptide sequences conserved in various taxonomic ranges using a novel pipeline. Esuca. BMC Genom. 21, 260 (2020).
Article CAS MATH Google Scholar
Sakiyama, K., Shimokawa-Chiba, N., Fujiwara, K. & Chiba, S. Search for translation arrest peptides encoded upstream of genes for components of protein localization pathways. Nucleic Acids Res. 49, 1550–1566 (2021).
Article CAS PubMed PubMed Central MATH Google Scholar
Fujiwara, K., Tsuji, N., Yoshida, M., Takada, H. & Chiba, S. Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery. Nat. Commun. 15, 2711 (2024).
Article ADS CAS PubMed PubMed Central Google Scholar
Imamichi, T. et al. Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species. Nucleic Acids Res. 52, 4276–4294 (2024).
Article CAS PubMed PubMed Central MATH Google Scholar
Meydan, S. et al. Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome. Mol. Cell 74, 481–493.e6 (2019).
Article CAS PubMed PubMed Central Google Scholar
Weaver, J., Mohammad, F., Buskirk, A. R. & Storz, G. Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes. mBio 10, e02819–18 (2019).
Article CAS PubMed PubMed Central Google Scholar
Stringer, A., Smith, C., Mangano, K. & Wade, J. T. Identification of Novel Translated Small Open Reading Frames in Escherichia coli Using Complementary Ribosome Profiling Approaches. J. Bacteriol. 204, e00352–21 (2021).
PubMed Google Scholar
Arenz, S. et al. Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide. Nat. Commun. 5, 3501 (2014).
Article ADS PubMed MATH Google Scholar
Sohmen, D. et al. Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling. Nat. Commun. 6, 6941 (2015).
Article ADS CAS PubMed MATH Google Scholar
Su, T. et al. The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling. Elife 6, e25642 (2017).
Article PubMed PubMed Central Google Scholar
Shanmuganathan, V. et al. Structural and mutational analysis of the ribosome-arresting human XBP1u. Elife 8, e46267 (2019).
Article PubMed PubMed Central MATH Google Scholar
Herrero Del Valle, A. et al. Ornithine capture by a translating ribosome controls bacterial polyamine synthesis. Nat. Microbiol. 5, 554–561 (2020).
Article CAS PubMed MATH Google Scholar
Beckert, B. et al. Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics. Nat. Commun. 12, 4466 (2021).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Fostier, C. R. et al. Regulation of the macrolide resistance ABC-F translation factor MsrD. Nat. Commun. 14, 3891 (2023).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Gersteuer, F. et al. The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome. Nat. Commun. 15, 2431 (2024).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Morici, M. et al. RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity. Nat. Commun. 15, 2432 (2024).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Gong, M., Gong, F. & Yanofsky, C. Overexpression of tnaC of Escherichia coli inhibits growth by depleting tRNA₂^Pro availability. J. Bacteriol. 188, 1892–1898 (2006).
Article CAS PubMed PubMed Central MATH Google Scholar
Chadani, Y. et al. Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing. Mol. Cell 68, 528–539.e5 (2017).
Article CAS PubMed Google Scholar
Hemm, M. R., Paul, B. J., Schneider, T. D., Storz, G. & Rudd, K. E. Small membrane proteins found by comparative genomics and ribosome binding site models. Mol. Microbiol. 70, 1487–1501 (2008).
Article CAS PubMed PubMed Central Google Scholar
VanOrsdel, C. E. et al. Identifying New Small Proteins in Escherichia coli. Proteomics 18, 1700064 (2018).
Article PubMed PubMed Central Google Scholar
Adams, P. P. et al. Regulatory roles of Escherichia coli 5’ UTR and ORF-internal RNAs detected by 3’ end mapping. eLife 10, e62438 (2021).
Article CAS PubMed PubMed Central Google Scholar
Michel, A. M. et al. GWIPS-viz: development of a ribo-seq genome browser. Nucleic Acids Res. 42, D859–D864 (2013).
Article PubMed PubMed Central Google Scholar
VanBogelen, R. A. & Neidhardt, F. C. Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc. Natl. Acad. Sci. Usa. 87, 5589–5593 (1990).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Jiang, W., Jones, P. & Inouye, M. Chloramphenicol induces the transcription of the major cold shock gene of Escherichia coli, cspA. J. Bacteriol. 175, 5824–5828 (1993).
Article CAS PubMed PubMed Central Google Scholar
Wilson, D. N. The A-Z of bacterial translation inhibitors. Crit. Rev. Biochem. Mol. Biol. 44, 393–433 (2009).
Article CAS PubMed MATH Google Scholar
Lin, J., Zhou, D., Steitz, T. A., Polikanov, Y. S. & Gagnon, M. G. Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design. Annu. Rev. Biochem. 87, 1–28 (2018).
Article Google Scholar
Lim, B., & Gross, C. A. Cellular response to heat shock and cold shock. In G. Storzand & R. Hengge (Eds.), Bacterial stress responses (2nd Ed., pp. 93-114). Washington, DC: ASM Press. (2011).
Goldstein, J., Pollitt, N. S. & Inouye, M. Major cold shock protein of Escherichia coli. Proc. Natl. Acad. Sci. Usa. 87, 283–287 (1990).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Jones, P. G., Mitta, M., Kim, Y., Jiang, W. & Inouye, M. Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli. Proc. Natl. Acad. Sci. Usa. 93, 76–80 (1996).
Article ADS CAS PubMed PubMed Central Google Scholar
Giuliodori, A. M., Brandi, A., Giangrossi, M., Gualerzi, C. O. & Pon, C. L. Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias. RNA 13, 1355–1365 (2007).
Article CAS PubMed PubMed Central Google Scholar
Kitagawa, M., Wada, C., Yoshioka, S. & Yura, T. Expression of ClpB, an analog of the ATP-dependent protease regulatory subunit in Escherichia coli, is controlled by a heat shock sigma factor (sigma 32). J. Bacteriol. 173, 4247–4253 (1991).
Article CAS PubMed PubMed Central Google Scholar
Miwa, T., Chadani, Y. & Taguchi, H. Escherichia coli small heat shock protein IbpA is an aggregation‐sensor that self‐regulates its own expression at posttranscriptional levels. Mol. Microbiol. 115, 142–156 (2021).
Article PubMed Google Scholar
Ito, Y. et al. Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes. Nat. Commun. 13, 7451 (2022).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Yamakawa, A., Niwa, T., Chadani, Y., Kobo, A. & Taguchi, H. A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell. Nucleic Acids Res. 51, e30–e30 (2023).
Article CAS PubMed PubMed Central Google Scholar
Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).
Article CAS PubMed MATH Google Scholar
Syroegin, E. A., Aleksandrova, E. V. & Polikanov, Y. S. Insights into the ribosome function from the structures of non-arrested ribosome–nascent chain complexes. Nat. Chem. 15, 143–153 (2023).
Article CAS PubMed Google Scholar
Zeng, F. & Jin, H. Conformation of methylated GGQ in the Peptidyl Transferase Center during Translation Termination. Sci. Rep. 8, 2349 (2018).
Article ADS PubMed PubMed Central MATH Google Scholar
Weiner, A. M. & Weber, K. Natural Read-through at the UGA Termination Signal of Qβ Coat Protein Cistron. Nat. N. Biol. 234, 206–209 (1971).
Article CAS Google Scholar
Engelberg-Kulka, H. UGA suppression by normal tRNA^TrP in Escherichia coli: codon context effects. Nucleic Acids Res. 9, 983–991 (1981).
Article CAS PubMed PubMed Central Google Scholar
Lovett, P. S., Ambulos, N. P., Mulbry, W., Noguchi, N. & Rogers, E. J. UGA can be decoded as tryptophan at low efficiency in Bacillus subtilis. J. Bacteriol. 173, 1810–1812 (1991).
Article CAS PubMed PubMed Central Google Scholar
Ramu, H. et al. Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center. Mol. Cell 41, 321–330 (2011).
Article CAS PubMed MATH Google Scholar
Chevance, F. F. V., Guyon, S. L. & Hughes, K. T. The Effects of Codon Context on In Vivo Translation Speed. PLoS Genetics 10, e1004392 (2014).
Article PubMed PubMed Central MATH Google Scholar
Verma, M. et al. A short translational ramp determines the efficiency of protein synthesis. Nat. Commun. 10, 5774 (2019).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Osterman, I. A. et al. Translation at first sight: the influence of leading codons. Nucleic Acids Res. 48, 6931–6942 (2020).
Article CAS PubMed PubMed Central MATH Google Scholar
Mottagui-Tabar, S., Björsson, A. & Isaksson, L. A. The second to last amino acid in the nascent peptide as a codon context determinant. EMBO J. 13, 249–257 (1994).
Article CAS PubMed PubMed Central Google Scholar
Björnsson, A., Mottagui-Tabar, S. & Isaksson, L. A. Structure of the C-terminal end of the nascent peptide influences translation termination. EMBO J. 15, 1696–1704 (1996).
Article PubMed PubMed Central Google Scholar
Sunohara, T., Abo, T., Inada, T. & Aiba, H. The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons. RNA 8, 1416–1427 (2002).
Article CAS PubMed PubMed Central Google Scholar
Hayes, C. S., Bose, B. & Sauer, R. T. Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination* 210. J. Biol. Chem. 277, 33825–33832 (2002).
Article CAS PubMed Google Scholar
van Nies, P. et al. Unbiased Tracking of the Progression of mRNA and Protein Synthesis in Bulk and in Liposome‐Confined Reactions. Chem. Bio. Chem. 14, 1963–1966 (2013).
Article PubMed MATH Google Scholar
Kazuta, Y., Matsuura, T., Ichihashi, N. & Yomo, T. Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system. J. Biosci. Bioeng. 118, 554–557 (2014).
Article CAS PubMed MATH Google Scholar
Matsuura, T., Tanimura, N., Hosoda, K., Yomo, T. & Shimizu, Y. Reaction dynamics analysis of a reconstituted Escherichia coli protein translation system by computational modeling. Proc. Natl. Acad. Sci. USA. 114, E1336–E1344 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Gruber, A. R., Lorenz, R., Bernhart, S. H., Neuböck, R. & Hofacker, I. L. The Vienna RNA Websuite. Nucleic Acids Res. 36, W70–W74 (2008).
Article CAS PubMed PubMed Central MATH Google Scholar
Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).
Article PubMed PubMed Central MATH Google Scholar
Chandu, D., Kumar, A. & Nandi, D. PepN, the Major Suc-LLVY-AMC-hydrolyzing Enzyme in Escherichia coli, Displays Functional Similarity with Downstream Processing Enzymes in Archaea and Eukarya IMPLICATIONS IN CYTOSOLIC PROTEIN DEGRADATION*. J. Biol. Chem. 278, 5548–5556 (2003).
Article CAS PubMed Google Scholar
Bhosale, M. et al. Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress. Microbiol. Res. 168, 56–64 (2013).
Article CAS PubMed MATH Google Scholar
Evers, S. et al. Mechanism‐related changes in the gene transcription and protein synthesis patterns of Haemophilus influenzae after treatment with transcriptional and translational inhibitors. Proteomics 1, 522–544 (2001).
Article CAS PubMed MATH Google Scholar
Bandow, J. E., Brötz, H., Leichert, L. I. O., Labischinski, H. & Hecker, M. Proteomic Approach to Understanding Antibiotic Action. Antimicrob. Agents Chemother. 47, 948–955 (2003).
Article CAS PubMed PubMed Central Google Scholar
Miller, J. H. Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1972).
Chiba, S. & Ito, K. Multisite Ribosomal Stalling: A Unique Mode of Regulatory Nascent Chain Action Revealed for MifM. Mol. Cell 47, 863–872 (2012).
Article CAS PubMed MATH Google Scholar
Onodera, H., Niwa, T., Taguchi, H. & Chadani, Y. Prophage excision switches the primary ribosome rescue pathway and rescue‐associated gene regulations in Escherichia coli. Mol. Microbiol. 119, 44–58 (2023).
Article CAS PubMed Google Scholar
Gillet, L. C. et al. Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis. Mol. Cell. Proteom. 11, O111.016717 (2012).
Article MATH Google Scholar
Demichev, V., Messner, C. B., Vernardis, S. I., Lilley, K. S. & Ralser, M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods 17, 41–44 (2020).
Article CAS PubMed Google Scholar
Midha, M. K. et al. A comprehensive spectral assay library to quantify the Escherichia coli proteome by DIA/SWATH-MS. Sci. Data 7, 389 (2020).
Article CAS PubMed PubMed Central Google Scholar
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Article CAS PubMed Google Scholar
Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).
Article CAS PubMed Google Scholar
Watson, Z. L. et al. Structure of the bacterial ribosome at 2 Å resolution. eLife 9, e60482 (2020).
Article CAS PubMed PubMed Central MATH Google Scholar
Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. Sect. D: Biol. Crystallogr. 71, 136–153 (2015).
Article ADS CAS MATH Google Scholar
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Article ADS PubMed MATH Google Scholar
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Article ADS CAS MATH Google Scholar
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Article ADS CAS PubMed MATH Google Scholar
Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).
Article CAS PubMed PubMed Central MATH Google Scholar
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Article CAS PubMed MATH Google Scholar
Okuda, S. et al. jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res. 45, D1107–D1111 (2017).
Article CAS PubMed MATH Google Scholar

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Acknowledgements

We thank Takashi Kanamori for providing customized PUREfrex v1.0, and the anonymous reviewers for their valuable comments. Additionally, we extend our thanks to the Integrative Bioscience Facility at the Institute of Science Tokyo for DNA sequencing, and the Cell Biology Center Research Core Facility at the Institute of Science Tokyo for the Q-Exactive mass spectrometry measurements. This work was supported by MEXT Grants-in-Aid for Scientific Research (Grant Number JP20H05925 to H.T.), Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP22ama121012 (support number 4857 to H.T.) and JP23ama121002 (support number 3273 to O.N.), Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Numbers 22H02553 to Y.I., 23H02410 to Y.C.), and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST) (Grant Number JPMJCR20E2 to O.N.), Pioneering Research Initiated by the Next Generation (SPRING) of Japan Science and Technology Agency (JST) (Grant Number JPMJSP2106 to A.K.), and by grants from the Ohsumi Frontier Science Foundation, the Japan Foundation for Applied Enzymology, the Takeda Science Foundation, the Yamada Science Foundation, and the Senri Life Science Foundation to Y.C.

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These authors contributed equally: Yushin Ando, Akinao Kobo, Tatsuya Niwa.

Authors and Affiliations

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
Yushin Ando, Osamu Nureki & Yuzuru Itoh
School of Life Science and Technology, Institute of Science Tokyo, Yokohama, Japan
Akinao Kobo, Tatsuya Niwa, Ayako Yamakawa, Suzuna Konoma, Yuki Kobayashi & Hideki Taguchi
Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Japan
Tatsuya Niwa & Hideki Taguchi
Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Okayama, Japan
Yuhei Chadani

Authors

Yushin Ando
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Akinao Kobo
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Tatsuya Niwa
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Ayako Yamakawa
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Suzuna Konoma
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Yuki Kobayashi
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Osamu Nureki
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Hideki Taguchi
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Yuzuru Itoh
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Yuhei Chadani
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Contributions

A.K., Y.C., S.K., and Y.K. performed genetic and biochemical experiments. T.N. and A.Y. performed mass-spectrometry measurements and data analyses. Y.A. collected the cryo-EM data and built the model. Y.A. and Y.I. analyzed the structure. Y.C., Y.A., A.K., T.N., Y.I., O.N., and H.T. wrote the manuscript. Y.C., Y.I., H.T., and O.N. supervised the research.

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Correspondence to Osamu Nureki, Hideki Taguchi, Yuzuru Itoh or Yuhei Chadani.

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Ando, Y., Kobo, A., Niwa, T. et al. A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism. Nat Commun 16, 2323 (2025). https://doi.org/10.1038/s41467-025-57659-z

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Received: 23 June 2024
Accepted: 25 February 2025
Published: 08 March 2025
DOI: https://doi.org/10.1038/s41467-025-57659-z