Early nascent polypeptide dynamics are coupled to the flexibility of the ribosomal tunnel constriction

The ribosomal exit tunnel, through which all nascent polypeptides emerge, is formed primarily by ribosomal RNA. Still, ribosomal proteins contribute roughly one quarter of the tunnel walls. In particular, proteins uL4 and uL22 define the narrowest region of the tunnel. This constriction, enriched in basic residues, mediates the earliest protein-protein contacts experienced by a nascent polypeptide. Here, we characterize its conformational dynamics by analyzing 222 Escherichia coli ribosome structures from the Protein Data Bank and by performing unbiased all-atom molecular dynamics simulations of the complete bacterial ribosome with nascent polypeptides of varying length and composition. In simulations of the empty ribosome, the constriction can transiently narrow below the diameter of a water molecule and thus fully occlude the tunnel. At other times it opens wide enough to accommodate a narrow -helix. The presence of even a short nascent polypeptide shifts the constriction toward wider conformations by approximately 0.2 nm, revealing an adaptive response of the tunnel to its contents. Across all simulated systems, the N-terminal formylmethionine preferentially associates with the tunnel wall at the uL22 side. Our findings replace the static picture of the tunnel constriction with a dynamic one in which the constriction site functions as a flexible gate, with implications for nascent polypeptide translocation, action of macrolide antibiotics, and the translational ramp.