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

Author:

Zhang Haolin1,Gong Xuemei12,Zhao Qianqian12,Mukai Takahito3ORCID,Vargas-Rodriguez Oscar3,Zhang Huiming12,Zhang Yuxing14,Wassel Paul5,Amikura Kazuaki3,Maupin-Furlow Julie56ORCID,Ren Yan1,Xu Xun1ORCID,Wolf Yuri I7ORCID,Makarova Kira S7ORCID,Koonin Eugene V7ORCID,Shen Yue189ORCID,Söll Dieter310ORCID,Fu Xian18ORCID

Affiliation:

1. BGI-Shenzhen, Shenzhen, 518083, China

2. 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. 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. 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. Guangdong Provincial Key Laboratory of Genome Read and Write, Shenzhen 518120, China

9. Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China

10. Department of Chemistry, Yale University, New Haven, CT 06511, USA

Abstract

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)/tRNAPyl 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/tRNAPyl 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/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl 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.

Funder

National Key Research and Development Program of China

National Natural Science Foundation of China

Natural Science Foundation of Guangdong Province

Guangdong Provincial Key Laboratory of Genome Read and Write

National Institute of General Medical Sciences

Department of Energy

NIH Intramural Research Program

Publisher

Oxford University Press (OUP)

Subject

Genetics

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