Altering the ribosome exit tunnel to improve consecutive incorporation of challenging monomers

Ribosomes are capable of incorporating a wide array of natural and unnatural monomers into growing polymer chains, but can be stalled by monomers with constrained or non-natural backbones. Here we evaluate whether monomer-dependent ribosome stalling can be alleviated by structure-guided mutations to 23S rRNA within the exit tunnel. Ribosomes harboring an A2062U mutation are as active as wild type (WT) ribosomes when translating non-proline sequences and up to 10-fold more active when translating sequences containing up to four consecutive proline residues. High-resolution cryo-EM structures of WT and A2062U mutant ribosomes containing a polyproline nascent chain reveal that the A2062U mutation relieves an exit tunnel constriction to better accommodate a conformationally restricted peptide chain. A2062U mutant ribosomes also improve translation of sequences containing multiple, consecutive β ² -hydroxy acids. These results provide a mechanistic basis for engineering the ribosome for improved translation of genetically encoded polymers with novel backbones.