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Abstract Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.
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Monitoring monomer-specific acyl–tRNA levels in cells with PARTI
Abstract We describe a new assay that reports directly on the acylation state of a user-chosen transfer RNA (tRNA) in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl–tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non-α-amino acid monomers into protein: (i) the origins of the apparent selectivity of translation with respect to β2-hydroxy acid enantiomers; (ii) the activity of PylRS variants for benzyl derivatives of malonic acid; and (iii) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2′,3′-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non-α-amino acid monomers into proteins.
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- Award ID(s):
- 2002182
- PAR ID:
- 10588596
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 53
- Issue:
- 8
- ISSN:
- 0305-1048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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