Title: The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism

Abstract

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNA^Pyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNA^Pyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNA^Pyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNA^Pyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNA^Pyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

Authors:

Zhang, Haolin ^[1]; Gong, Xuemei ^[2]; Zhao, Qianqian ^[2]; Mukai, Takahito  ^[3];

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Vargas-Rodriguez, Oscar ^[3]; Zhang, Huiming ^[2]; Zhang, Yuxing ^[4]; Wassel, Paul ^[5]; Amikura, Kazuaki ^[3]; Maupin-Furlow, Julie  ^[6];

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Ren, Yan ^[1]; Xu, Xun  ^[1];

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Wolf, Yuri I.  ^[7];

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Makarova, Kira S.  ^[7];

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Koonin, Eugene V.  ^[7];

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Shen, Yue  ^[8];

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Söll, Dieter  ^[9];

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Fu, Xian  ^[10]

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• Search DOE PAGES for ORCID "0000-0001-9044-8850"

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+ Show Author Affiliations

1. BGI-Shenzhen, Shenzhen, 518083, China
2. BGI-Shenzhen, Shenzhen, 518083, China, College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China
3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA
4. BGI-Shenzhen, Shenzhen, 518083, China, Sino-Danish College, University of the Chinese Academy of Sciences, Beijing, China
5. Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA
6. Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA, Genetics Institute, University of Florida, Gainesville, FL 32611, USA
7. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA
8. BGI-Shenzhen, Shenzhen, 518083, China, Guangdong Provincial Key Laboratory of Genome Read and Write, Shenzhen 518120, China, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
9. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA, Department of Chemistry, Yale University, New Haven, CT 06511, USA
10. BGI-Shenzhen, Shenzhen, 518083, China, Guangdong Provincial Key Laboratory of Genome Read and Write, Shenzhen 518120, China

Publication Date:

Mon Apr 25 00:00:00 EDT 2022

Research Org.:

Univ. of Florida, Gainesville, FL (United States); Yale Univ., New Haven, CT (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES); National Key Research and Development Program of China; National Natural Science Foundation of China (NSFC); Natural Science Foundation of Guangdong Province; Guangdong Provincial Key Laboratory of Genome Read and Write; National Institute of General Medical Sciences; National Institutes of Health (NIH)

OSTI Identifier:

1864606

Alternate Identifier(s):

OSTI ID: 1904664

Grant/Contract Number:  

FG02-98ER20311; FG02-05ER15650; 2018YFA0900100; 31901029; 31800078; 2021A1515010995; 2017B030301011; R35GM122560; 3R35GM122560-05S1; R01GM057498

Resource Type:

Published Article

Journal Name:

Nucleic Acids Research

Additional Journal Information:

Journal Name: Nucleic Acids Research Journal Volume: 50 Journal Issue: 8; Journal ID: ISSN 0305-1048

Publisher:

Oxford University Press

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES