Escherichia coli ribosomes support translation of (R) and (S) β2-hydroxyacids in vitro: a structural and biochemical study

The ribosomal incorporation of backbone-modified amino acid analogs into peptides and proteins enables the programmed synthesis of sequence-defined biopolymers with tunable properties. However, the successful use of backbone-modified monomers as substrates by the ribosome requires coordination across multiple parts of the translation machinery, including aminoacyl-tRNA synthetases, translation factors, and finally the ribosome itself. β-hydroxyacids are particularly interesting monomers because they have potential to support the programmed biosynthesis of both polyesters (plastics) and depsipeptides (therapeutics). Previous work has reported that both enantiomers of β2-hydroxy-Nε-Boc-Lysine (β2-OH-BocK) are in vitro substrates for the orthogonal M. alvi PylRS/tRNA pair, but only one enantiomer is introduced into protein in vivo and with substantially lower yield than expected. We sought to determine whether there is a structural basis for the diminished yield as well as the preferential incorporation of one β2-OH-BocK enantiomer over the other. Here we report high-resolution cryo-EM structures of the Escherichia coli (E. coli) ribosome complexed with either (R)- or (S)-β2-OH-BocK. These structures reveal that both enantiomers are well positioned to undergo bond formation within the ribosome active site and are likely equally reactive. In vitro translation experiments confirm that orthogonal tRNAs acylated with (R)- or (S)-β2-OH-BocK are ribosome substrates, implying that the preferential incorporation of one enantiomer over the other in vivo results from deficiencies in other translation steps, such as tRNA acylation efficiency in cells or delivery to the ribosome by elongation factor Tu (EF-Tu). Taken together, this work demonstrates the plasticity of the E. coli ribosome and its tolerance for diverse substrates.