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Synthesis of Peptidyl-tRNA Mimics for Structural Biology Applications
[Image: see text] Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine—the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRN...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10552525/ https://www.ncbi.nlm.nih.gov/pubmed/37728742 http://dx.doi.org/10.1021/acs.accounts.3c00412 |
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author | Polikanov, Yury S. Etheve-Quelquejeu, Mélanie Micura, Ronald |
author_facet | Polikanov, Yury S. Etheve-Quelquejeu, Mélanie Micura, Ronald |
author_sort | Polikanov, Yury S. |
collection | PubMed |
description | [Image: see text] Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine—the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry. Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule–NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis. Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs. In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3′-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging. Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis. |
format | Online Article Text |
id | pubmed-10552525 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-105525252023-10-06 Synthesis of Peptidyl-tRNA Mimics for Structural Biology Applications Polikanov, Yury S. Etheve-Quelquejeu, Mélanie Micura, Ronald Acc Chem Res [Image: see text] Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine—the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry. Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule–NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis. Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs. In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3′-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging. Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis. American Chemical Society 2023-09-20 /pmc/articles/PMC10552525/ /pubmed/37728742 http://dx.doi.org/10.1021/acs.accounts.3c00412 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Polikanov, Yury S. Etheve-Quelquejeu, Mélanie Micura, Ronald Synthesis of Peptidyl-tRNA Mimics for Structural Biology Applications |
title | Synthesis of
Peptidyl-tRNA Mimics for Structural Biology
Applications |
title_full | Synthesis of
Peptidyl-tRNA Mimics for Structural Biology
Applications |
title_fullStr | Synthesis of
Peptidyl-tRNA Mimics for Structural Biology
Applications |
title_full_unstemmed | Synthesis of
Peptidyl-tRNA Mimics for Structural Biology
Applications |
title_short | Synthesis of
Peptidyl-tRNA Mimics for Structural Biology
Applications |
title_sort | synthesis of
peptidyl-trna mimics for structural biology
applications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10552525/ https://www.ncbi.nlm.nih.gov/pubmed/37728742 http://dx.doi.org/10.1021/acs.accounts.3c00412 |
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