Evolution of the ribosomal exit tunnel through the eyes of the nascent chain

Abstract

The exit tunnel is a universally conserved feature of the ribosomal large subunit that directs the nascent polypeptide chain into the cellular environment and is involved in co-translational folding, stalling, and antibiotic binding. While recent advances in cryo-EM have revealed organism-specific variations in ribosome structure, tunnel definition and comparative analyses have largely relied on pure geometric algorithms that often approximate the tunnel as a static, single tube with varying radius. Here, we present a functional, nascent-chain-centric characterisation of the exit tunnel across the tree of life, derived from all-atom molecular dynamics simulations using the most complete set of 55 distinct cytoplasmic ribosome structures. By mapping the steric accessibility through the "eyes" of the nascent chain (NC) at five different stages of translation, we reveal a topological and stage-dependent complexity invisible to static geometric approaches, demonstrating how tunnel accessibility dynamically changes during biosynthesis. We identify transiently accessible, bacteria-specific lateral branches ("side tunnels") that effectively expand the functional volume of the tunnel but may also serve as kinetic traps for the nascent chain. In contrast, we show that archaeal and eukaryotic ribosomes have structurally occluded these cavities by incorporating the eL39 protein. These evolutionary "plugs" seal the tunnel wall and decrease its functional width. Collectively, our results demonstrate that the ribosome exit tunnel is not a simple linear tube, but a branched, lineage-specific landscape where accessibility is temporally gated by nascent chain length. This functional definition provides a new framework for understanding how ribosomal architecture modulates the early stages of protein biogenesis.