Uncovering translation roadblocks during the development of a synthetic tRNA
Abstract
Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
- Authors:
-
- Stanford Univ., CA (United States)
- Yale Univ., New Haven, CT (United States)
- Columbia Univ., New York, NY (United States)
- Uppsala Univ. (Sweden)
- Publication Date:
- Research Org.:
- Yale Univ., New Haven, CT (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); Cystic Fibrosis Foundation; Knut and Alice Wallenberg Foundation
- OSTI Identifier:
- 1904746
- Grant/Contract Number:
- FG02-98ER20311; R35 GM122560; R35 GM122560-05S1; GM51266; AI15046; T32-GM008294; PUGLIS20G0; KAW 2016.0488
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 50; Journal Issue: 18; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Biochemistry & Molecular Biology
Citation Formats
Prabhakar, Arjun, Krahn, Natalie, Zhang, Jingji, Vargas-Rodriguez, Oscar, Krupkin, Miri, Fu, Ziao, Acosta-Reyes, Francisco J., Ge, Xueliang, Choi, Junhong, Crnković, Ana, Ehrenberg, Måns, Puglisi, Elisabetta Viani, Söll, Dieter, and Puglisi, Joseph. Uncovering translation roadblocks during the development of a synthetic tRNA. United States: N. p., 2022.
Web. doi:10.1093/nar/gkac576.
Prabhakar, Arjun, Krahn, Natalie, Zhang, Jingji, Vargas-Rodriguez, Oscar, Krupkin, Miri, Fu, Ziao, Acosta-Reyes, Francisco J., Ge, Xueliang, Choi, Junhong, Crnković, Ana, Ehrenberg, Måns, Puglisi, Elisabetta Viani, Söll, Dieter, & Puglisi, Joseph. Uncovering translation roadblocks during the development of a synthetic tRNA. United States. https://doi.org/10.1093/nar/gkac576
Prabhakar, Arjun, Krahn, Natalie, Zhang, Jingji, Vargas-Rodriguez, Oscar, Krupkin, Miri, Fu, Ziao, Acosta-Reyes, Francisco J., Ge, Xueliang, Choi, Junhong, Crnković, Ana, Ehrenberg, Måns, Puglisi, Elisabetta Viani, Söll, Dieter, and Puglisi, Joseph. Wed .
"Uncovering translation roadblocks during the development of a synthetic tRNA". United States. https://doi.org/10.1093/nar/gkac576. https://www.osti.gov/servlets/purl/1904746.
@article{osti_1904746,
title = {Uncovering translation roadblocks during the development of a synthetic tRNA},
author = {Prabhakar, Arjun and Krahn, Natalie and Zhang, Jingji and Vargas-Rodriguez, Oscar and Krupkin, Miri and Fu, Ziao and Acosta-Reyes, Francisco J. and Ge, Xueliang and Choi, Junhong and Crnković, Ana and Ehrenberg, Måns and Puglisi, Elisabetta Viani and Söll, Dieter and Puglisi, Joseph},
abstractNote = {Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.},
doi = {10.1093/nar/gkac576},
journal = {Nucleic Acids Research},
number = 18,
volume = 50,
place = {United States},
year = {Wed Jul 27 00:00:00 EDT 2022},
month = {Wed Jul 27 00:00:00 EDT 2022}
}
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