Unveiling a Novel Translation Arrest Mechanism - The Mini-Hairpin Peptide That Defies Ribosome Termination

The intricate dance of protein synthesis, where ribosomes decode genetic information into functional proteins, is tightly regulated by molecular checkpoints. While translation termination marks the final act of this process, recent discoveries reveal unexpected players that can halt this machinery with precision. A groundbreaking study published in Nature Communications (2025)¹ uncovers how a nascent peptide, PepNL, adopts a unique structural configuration to block termination, offering fresh insights into ribosomal regulation.

Translation Termination and the Enigma of Ribosome Arrest

Protein synthesis culminates in translation termination, a tightly regulated process where release factors (RFs) recognize stop codons to hydrolyze the bond between the nascent peptide and tRNA. In bacteria, RF1 and RF2 mediate this step by inserting their conserved GGQ motif into the peptidyl transferase center (PTC) of the ribosome. However, certain nascent peptides disrupt this process by interacting with the ribosome exit tunnel, a phenomenon termed translation arrest.

The nascent peptide exit tunnel (NPET), lined with negatively charged rRNA and ribosomal proteins, serves as a conduit for elongating polypeptides. Specific amino acid sequences within nascent peptides can form interactions with NPET components, stalling ribosomes mid-translation. Classic examples include E. coli TnaC, which arrests translation in response to high tryptophan levels, and SecM, which regulates downstream gene expression by sensing mechanical force. Despite advances, the diversity of arrest mechanisms and their regulatory roles remain poorly understood.

Screening for Hidden Arrest Peptides: The Emergence of PepNL and NanCL

To uncover novel arrest peptides, researchers systematically screened E. coli small open reading frames (sORFs) using phenotypic assays, proteomics, and mass spectrometry. Overexpression of known arrest peptides like TnaC and SecM inhibits bacterial growth by sequestering ribosomes, a hallmark of translation arrest. Applying this principle to 38 uncharacterized sORFs, two candidates—PepNL and NanCL—stood out for their ability to induce growth inhibition and cold shock protein (CSP) expression, a signature of stalled translation.

Proteomic profiling revealed that PepNL overexpression triggered a stress response akin to translation inhibitors like chloramphenicol. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptidyl-tRNA intermediates confirmed ribosome stalling at PepNL's UGA stop codon. Frameshift mutations in pepNL abolished both cytotoxicity and stalling, validating its role as a bona fide arrest peptide. Intriguingly, pepNL is positioned upstream of pepN, a gene encoding an aminopeptidase, suggesting regulatory crosstalk between translation arrest and metabolic adaptation.

Cryo-EM Reveals a Mini-Hairpin Fold That Blocks Release Factor Access

The structural basis of PepNL's arrest activity was unraveled through cryo-electron microscopy (cryo-EM). Unlike typical arrest peptides that extend toward the tunnel exit, PepNL adopts a mini-hairpin conformation, folding its N-terminal residues back toward the PTC. This compact structure, stabilized by hydrophobic interactions between Ile3, Leu4, Ile8, and Tyr9, distorts the C-terminal region of the peptide. The distortion forces Ile13 into a steric clash with the GGQ motif of RF2, preventing its proper positioning in the PTC.

Key Structural Features of PepNL:

1. Intramolecular Interactions: A β-sheet-like network between Lys2-Ala10 and Leu4-Ile8 stabilizes the hairpin.
2. rRNA Engagement: Hydrophobic contacts with 23S rRNA nucleotides (e.g., U2609, A2058) anchor the peptide within the tunnel.
3. RF2 Rearrangement: The clash with Ile13 triggers a 16-Å displacement of RF2's Gln252, rendering the GGQ motif inactive.

This mechanism contrasts sharply with sensory arrest peptides like TnaC, which rely on inducers (e.g., tryptophan) to alter ribosomal architecture. PepNL's sequence-dependent stalling operates independently of external signals, highlighting its uniqueness.

Fig 1 The versatile face of the β-hairpin scaffold XWXWXpPXK(/R)X(R).²

Tryptophan as an Arrest Inhibitor: A Regulatory Paradox

While most arrest peptides require inducers to activate stalling, PepNL flips this paradigm. Instead of needing an inducer, its arrest is inhibited by tryptophan. Depleting tryptophan from in vitro translation systems robustly stalled ribosomes at PepNL's UGA codon. However, adding tryptophan enabled Trp-tRNATrp to read through the stop codon, bypassing arrest.

Mechanistic Insights:

• Stop Codon Specificity: PepNL's UGA codon is uniquely susceptible to read-through by Trp-tRNATrp, unlike UAA or UAG.
• Kinetic Competition: The hairpin structure forms post-translationally, allowing Trp-tRNATrp to act before steric clashes arise.
• Dual Inhibition: Once the hairpin forms, it impedes both RF2-mediated termination and further elongation, slowing ribosome progression to ~1 amino acid per minute.

This regulatory twist positions tryptophan as a metabolic rheostat, linking nutrient availability to translational control. Under tryptophan-rich conditions, read-through prevents ribosome sequestration, ensuring PepN expression for amino acid recycling—a survival advantage in fluctuating environments.

Broader Implications: Rewriting the Rules of Translation Control

The discovery of PepNL's mini-hairpin mechanism expands the repertoire of ribosome arrest strategies. Its ability to stall without inducers suggests that nascent peptides may exploit intrinsic structural dynamics to regulate translation. This challenges the dogma that arrest peptides universally require external triggers, highlighting the diversity of translational regulation.

Future Directions:

1. Evolutionary Conservation: Are similar mini-hairpin peptides present in eukaryotes or pathogenic bacteria?
2. Therapeutic Potential: Could synthetic arrest peptides be engineered to modulate translation in disease contexts?
3. Ribosome Profiling: High-resolution studies may uncover hidden arrest peptides in non-canonical ORFs.

Moreover, the interplay between PepNL and pepN hints at a feedback loop where translational stalling fine-tunes metabolic enzyme production. Such systems-level regulation could inspire synthetic biology applications, such as designing biosensors or stress-responsive circuits.

Conclusion: Decoding the Language of Nascent Peptides

The ribosome, once viewed as an indiscriminate protein factory, is increasingly recognized as a dynamic sensor of nascent peptide sequences. PepNL's mini-hairpin mechanism exemplifies how short peptides can exert profound regulatory effects through precise structural interactions. By blocking RF2 access and leveraging tryptophan as an inhibitor, PepNL exemplifies nature's ingenuity in balancing protein synthesis with metabolic demands.

As cryo-EM and omics technologies advance, the hidden world of regulatory peptides will continue to emerge. Each discovery, like PepNL, not only solves a molecular puzzle but also redefines our understanding of life's intricate regulatory networks. In the evolving narrative of translation control, nascent peptides are proving to be both authors and editors, scripting their own roles in cellular survival.

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References

1. Ando, Yushin, et al. "A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism." Nature Communications 16.1 (2025): 2323. https://doi.org/10.1038/s41467-025-57659-z
2. Stanojlovic, Vesna, et al. "A Conformationally Stable Acyclic β‐Hairpin Scaffold Tolerating the Incorporation of Poorly β‐Sheet‐Prone Amino Acids." ChemBioChem 23.4 (2022): e202100604. https://doi.org/10.1002/cbic.202100604. Distributed under the Open Access license CC BY 4.0, without modification.