# Molecular architecture and diversity of StopGo/2A translational recoding

## TL;DR

This paper reveals the molecular mechanism of StopGo/2A translational recoding and identifies sequence variations that could improve its use in biotechnology.

## Contribution

The study provides the first cryo-EM structure of a ribosome at a StopGo site and expands the known sequence motifs for functional StopGo elements.

## Key findings

- Cryo-EM structure shows how F2A interactions with the ribosome induce conformational changes that enable StopGo.
- Bioinformatic analysis identified expanded StopGo motifs and rare functional variants across nearly 10,000 viral sequences.
- No evidence of natural StopGo use by plant viruses was found despite its activity in plants.

## Abstract

StopGo is a genetic recoding strategy that cotranslationally produces two sequentially encoded proteins from a single open reading frame. StopGo elements are extensively used for coexpression of multiple genes in biotechnology, agriculture, and biomedicine, enabling efficient production of high-value therapeutic proteins, generation of pluripotent stem cells and usage in cancer therapies. Here, we determined the cryo-EM structure of a mammalian ribosome stalled at the foot-and-mouth disease virus StopGo (F2A) site, revealing how nascent peptide-exit tunnel interactions remodel the catalytic center. Combined with comprehensive analysis of 2A-peptide sequence diversity and host associations, our findings advance understanding of translational recoding and highlight ways in which StopGo could be optimized for superior multigene expression or employed as a potential target for antiviral drugs.

Viral 2A sequences trigger a cotranslational peptide bond formation “skipping” event, termed “StopGo,” to generate two separate proteins from a single open reading frame without classical termination. To investigate the mechanism of StopGo, we determined the cryo-EM structure of a mammalian ribosome positioned at the foot-and-mouth disease virus 2A (F2A) site. The structure shows how interactions between the F2A nascent chain (NC) and the ribosomal exit tunnel induce a conformational change in the peptidyl transferase center that precludes further translation elongation but instead pre-exposes the P-tRNA:F2A-NC ester bond for hydrolysis and NC release. Additionally, we bioinformatically characterized variation and host association across nearly 10,000 StopGo sequences identified in virus genomes. We expanded the canonical core motif to (D/G/C/N)(V/I)ExNPGP and identified additional rare but functional variants. We also revealed several distinct upstream motifs that we showed biochemically to be important for StopGo activity. Interestingly, although StopGo is known to be functionally active in plants, we found no evidence for natural utilization of StopGo by plant viruses. Overall, these findings provide valuable insights into a unique translation recoding mechanism, and lay foundations for further optimization of multigene expression in biotechnology.