9o2x
Structure of WT E.coli ribosome 70S subunit with complexed with mRNA, P-site fMet-NH-tRNAfMet and A-site (S)-betahydroxyBocK charged NH-tRNAPyl
Structural highlights
Publication Abstract from PubMedEngineering the translation apparatus to accept backbone-modified amino acid analogues would enable the programmed synthesis of sequence-defined biopolymers with tunable properties. beta-Hydroxy acids are of particular interest because they could support the programmed biosynthesis of both biocompatible polyester materials as well as natural product-like depsipeptides. Previous work has reported that both enantiomers of beta(2)-hydroxy-N(epsilon)-Boc-lysine (beta(2)-OH-BocK) are in vitro substrates for the orthogonal M. alvi pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair, but only one enantiomer is introduced into protein in vivo. Here we make use of high-resolution cryogenic electron microscopy (cryo-EM) to determine the structural basis for this observation. These structures reveal both beta(2)-OH-BocK isomers equally well-positioned within the ribosomal A site regardless of stereochemistry. Consistent with this observation, in vitro translation reactions charged with tRNAs acylated with (R)- or (S)-beta(2)-OH-BocK produced roughly equal amounts of translated product when quantified on the basis of either mass spectrometry or luminescence. Together, these experiments imply that the substantial preferential in vivo incorporation of one enantiomer over the other observed previously results primarily from deficiencies in the steps that precede bond formation by the E. coli ribosome. Indeed, as predicted by this work and demonstrated in an accompanying paper (Soni, C. Co-Translational Incorporation of (R)- and (S)-beta(2)-Hydroxyacids In Vivo: Directed Evolution of Efficient Aminoacyl-tRNA Synthetases. J. Am. Chem. Soc. 2026, 148, 10.1021/jacs.5c18595), when cells are provided with an active and orthogonal aminoacyl-tRNA synthetase/tRNA pair that accepts both (R)- and (S)-beta(2)-OH-BocK as substrates, both monomers are introduced into protein in good yield and with high fidelity. Co-Translational Incorporation of (R)- and (S)-beta(2)-Hydroxy Acids In Vitro: A Structural and Biochemical Study on the E. coli Ribosome.,Majumdar C, Kent AD, Hamlish NX, Zhu C, Fitzgerald KA, Cate JHD, Schepartz A J Am Chem Soc. 2026 Mar 11;148(9):9393-9399. doi: 10.1021/jacs.5c18603. Epub 2026 , Feb 27. PMID:41757712[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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