tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut

Author:

Krahn Natalie1ORCID,Zhang Jingji2,Melnikov Sergey V3,Tharp Jeffery M1,Villa Alessandra4,Patel Armaan1,Howard Rebecca J5,Gabir Haben6,Patel Trushar R789ORCID,Stetefeld Jörg610,Puglisi Joseph2ORCID,Söll Dieter111ORCID

Affiliation:

1. Department of Molecular Biophysics and Biochemistry, Yale University , New Haven , CT 06520 , USA

2. Department of Structural Biology, Stanford University School of Medicine , Stanford , CA  94305 , USA

3. Biosciences Institute, Newcastle University , Newcastle upon Tyne , NE2 4HH, UK

4. PDC-Center for High Performance Computing, KTH-Royal Institute of Technology , Stockholm , SE-100 44, Sweden

5. Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University , Solna , SE-171 65, Sweden

6. Department of Chemistry, University of Manitoba , Winnipeg , MB R3T 2N2 , Canada

7. Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge , Lethbridge , AB T1K 2E1 , Canada

8. Li Ka Shing Institute of Virology, University of Alberta , Edmonton , AB T6G 2E1 , Canada

9. Department of Microbiology, Immunology & Infectious Diseases, Cumming School of Medicine, University of Calgary , Calgary , AB T2N 4N1 , Canada

10. Department of Microbiology, University of Manitoba , Winnipeg , MB R3T 2N2 , Canada

11. Department of Chemistry, Yale University , New Haven , CT 06520 , USA

Abstract

Abstract Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G–U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G–C base pairs (G3:C70 and G5:C68).

Funder

NIH

Department of Energy Office of Basic Energy Sciences

Cystic Fibrosis Foundation

Canadian Institutes of Health Research

The Knut and Alice Wallenberg Foundation

Diamond Light Source

Tier-1 Canada Research Chair in Structural Biology

Publisher

Oxford University Press (OUP)

Subject

Genetics

Reference47 articles.

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3. Identity elements of tRNA as derived from information analysis;Zamudio;Orig. Life Evol. Biosph.,2018

4. Ancestral archaea expanded the genetic code with pyrrolysine;Guo;J. Biol. Chem.,2022

5. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool;Wan;Biochim. Biophys. Acta,2014

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