Abstract
multiple genetic codes developed during the evolution of eukaryotes and bacteria, yet no alternative genetic code is known for archaea. We used proteomics to confirm our prediction that certain archaea consistently incorporate pyrrolysine (Pyl) at TAG codons, supporting an alternative archaeal genetic code that we designate the Pyl code. This genetic code has 62 sense codons encoding 21 amino acids. In contrast to monophyletic genetic code distributions in bacteria, the archaeal Pyl code occurs sporadically, indicating that it arose independently in multiple lineages. We discovered that more than 1800 archaeal proteins contain Pyl, increasing the number of such proteins by two orders of magnitude. Additionally, five Pyl transfer rNA (trNA) pyrrolysyl–trNA synthetase pairs from Pyl-code archaea were used to introduce Pyl analogs into proteins in Escherichia coli.
| Original language | English |
|---|---|
| Article number | eadu2404 |
| Journal | Science |
| Volume | 390 |
| Issue number | 6775 |
| DOIs | |
| State | Published - Nov 20 2025 |
Funding
We thank J.-F. Brugere, J. Rylee, and P. Penev for helpful discussions. S.G. wishes to thank the Miller Institute at UC Berkeley for a Visiting Professorship. This work was supported by a Tory Burch Foundation Fellowship to V.K. at the Innovative Genomics Institute. This work used Bridges-2 at the Pittsburgh Supercomputing Center through allocation BIO230230 to V.K., from the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program, which is supported by National Science Foundation (NSF) grant nos. 2138259, 2138286, 2138307, 2137603, and 2138296. L.T.R. was supported by the NSF Graduate Research Fellowship Program (DGE-1752814). Experimental validation of PylRS activity for α-amino and α-hydroxy acids was supported by the NSF Center for Genetically Encoded Materials (C-GEM; CHE 2002182). Funding was provided by the Chan-Zuckerberg Initiative Foundation, Technology Enabled Biological Carbon Capture and Sequestration (CZIF2022-007203), and NSF Collaborative Research: TRTech-PGR Track (2334028) to J.F.B. G.B. was supported by Agence Nationale de la Recherche (ANR-19-CE02-0005). Research in S.G.’s laboratory is supported by the Fondation pour la Recherche Médicale (Programme equipes FRM EQU202203014614). Proteome work done at Oak Ridge National Laboratory was supported by a US Department of Energy Genome Sciences Program project (ERKPA50). We thank J.-F. Brugere,J. Rylee, and P. Penev for helpful discussions. S.G. wishes to thank the Miller Institute at UC Berkeley for a Visiting Professorship. Funding: This work was supported by a Tory Burch Foundation Fellowship to V.K. at the Innovative Genomics Institute.This work used Bridges-2 at the Pittsburgh Supercomputing Center through allocation BIO230230 to V.K., from the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program, which is supported by National Science Foundation (NSF) grant nos. 2138259, 2138286, 2138307, 2137603, and 2138296. L.T.R. was supported by the NSF Graduate Research Fellowship Program (DGE-1752814). Experimental validation of PylRS activity for ⍺-amino and ⍺-hydroxy acids was supported by the NSF Center for Genetically Encoded Materials (C-GEM; CHE 2002182). Funding was provided by the Chan-Zuckerberg Initiative Foundation,Technology Enabled Biological Carbon Capture and Sequestration (CZIF2022-007203), and NSF Collaborative Research: TRTech-PGR Track (2334028) to J.F.B. G.B. was supported by Agence Nationale de la Recherche (ANR-19-CE02-0005). Research in S.G.’s laboratory is supported by the Fondation pour la Recherche Médicale (Programme equipes FRM EQU202203014614). Proteome work done at Oak Ridge National Laboratory was supported by a US Department of Energy Genome Sciences Program project (ERKPA50). Author contributions: V.K. and J.F.B. conceived and designed the study.V.K. performed codon analysis, identification of Pyl proteins, and anaerobic cultivation of M. burtonii. G.B. prepared the genome database and identified Pyl machinery proteins and tRNA. Experimental validation of PylRS activity for ⍺-amino and ⍺-hydroxy acids was performed by N.X.H. and L.T.R. with oversight by A.S. Proteomics experiments and data analysis were conducted by S.L.P. with oversight by R.L.H. Phylogenetic analysis and discussion were conducted by G.B. and S.G. Structural prediction of Pyl-containing proteins and bioinformatics software development were performed by A.K.Anaerobic cultivation of M. alvus was performed by K.F. with oversight by G.B.The manuscript was written by V.K. and J.F.B. with input from all authors.All authors read and approved the manuscript. Competing interests: V.K., L.T.R., N.X.H.,A.S., and J.F.B. are inventors on provisional patent application 63/698,833 submitted by the University of California, Berkeley, that covers uses of this research. Data and materials availability: Proteomic data from this study have been deposited into the ProteomeXchange repository with accession nos. ProteomeXchange-PXD053523 and MassIVE-MSV000095193.The modified version of Prodigal with the Pyl code that has TAG reassigned to Pyl and the python script (find_tag_end.py) can be found on Zenodo (86). License information: Copyright © 2025 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. https://www.science.org/about/science-licenses-journal-article-reuse. This research was funded in whole or in part by Agence Nationale de la Recherche (ANR-19-CE02-0005), a cOAlition S organization.The author will make the Author Accepted Manuscript (AAM) version available under a CC BY public copyright license.
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