Ribosome dynamics at the conserved PGP motif controls 2A peptide-bond-skipping efficiency

Despite several decades of discovery, the molecular details of 2A-mediated peptide bond skipping remains incompletely understood. Here, we combine quantitative 2A reporters with high resolution ribosome profiling to monitor ribosome dynamics at the core 2A sequences.

We find that ribosome pausing at the last proline codon of the PGP motif serves as a kinetic decision point, and that the dwell time at the terminal proline inversely correlates with 2A skipping efficiency. Inserting a flexible linker immediately upstream of the 2A sequence reduces ribosome occupancy at the terminal proline codon, resulting in an enhanced peptide bond skipping. Introducing amino acid repeats into a distal upstream region also modulates the 2A activity, suggesting a long-range communication between nascent chains outside of the ribosome and inside the peptidyl transferase center.