Oral Presentation Australasian RNA Biology and Biotechnology Association 2026 Conference

Engineering the initiator tRNA: understanding and manipulating the anticodon identity set (144177)

Dominic Scopelliti 1 2 , Russel M Vincent 3 4 , Andras Hutvagner 1 2 , Fiona Whelan 5 , William P Klare 6 , Ignatius Peng 6 , George M Church 3 4 , Paul R Jaschke 1 2
  1. ARC Centre of Excellence in Synthetic Biology, Macquarie University, Macquarie Park, NSW, Australia
  2. School of Natural Sciences, Macquarie University, Macquarie Park, NSW, Australia
  3. Department of Genetics, Harvard Medical School, Boston, MA, The United States of America
  4. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, The United States of America
  5. Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia
  6. Australian Proteome Analysis Facility, Macquarie University, Macquarie Park, NSW, Australia

Orthogonal translation initiation offers a route to reprogram protein synthesis, expand the genetic code, and insulate synthetic circuits from native translation [1]. In bacteria, initiation depends on pairing between the initiator tRNA (i-tRNA) anticodon and a start codon downstream of the Shine-Dalgarno sequence. We asked how far the bacterial i-tRNA anticodon can be reprogrammed to create predictable start-codon identities, and how anticodon changes connect codon recognition, amino acid identity, and host physiology.

We first defined the complete i-tRNA anticodon identity set by comparing all 63 non-canonical anticodon mutants against GFP reporters bearing all 64 possible start codons [2–4]. These 4,096 combinations revealed a constrained but structured landscape: 20 mutant i-tRNAs initiated efficiently from complementary codons, nine were orthogonal, and non-complementary initiation was rare (<1% of pairs) [3]. To connect codon recognition with amino acid identity, we purified reporter proteins initiated by the top 31 i-tRNA–start-codon pairs and identified their N-terminal amino acids by data-dependent LC-MS/MS. Of these, 29 yielded identifiable N-terminal peptides, showing that anticodon reprogramming often redirects aminoacylation: many i-tRNAs were charged according to their new anticodon identity, and diverse non-methionyl amino acids were incorporated, frequently in formylated form [4].

We next examined how an orthogonal initiation system affects host physiology, focusing on a valine-incorporating i-tRNA-AAC mutant that initiates at GUU. Using fluorescence assays, proteomics, and tRNA sequencing, we identified two dominant bottlenecks—aminoacylation by valyl-tRNA synthetase (ValRS) and formylation by methionyl-tRNA formyltransferase (FMT). Overexpressing both enzymes improved efficiency and fidelity, but perturbed endogenous tRNA aminoacylation and acp3U47 modification, and triggered proteome-wide metabolic remodelling [5].

Finally, we tested portability by expressing a 12-member library of anticodon-mutant i-tRNAs paired with complementary non-canonical start codons across six common E. coli laboratory strains [6]. Non-canonical initiation was broadly functional but strongly host-dependent: inducibility, output, and fitness varied by strain, with comparative genomics linking this variation to strain-specific gene content and endogenous tRNA sequence and copy number. In probiotic Nissle 1917, some non-canonical codons exceeded canonical AUG output [6], highlighting this strain as a promising chassis for future microbiome-focused applications of engineered i-tRNAs.

Together, this work defines the i-tRNA anticodon identity set as a tuneable but interdependent design space in which codon recognition, amino acid identity, charging efficiency, and host compatibility must be co-optimised for robust orthogonal translation initiation.

  1. Liu, C., Jewett, M., Chin, J. et al. Toward an orthogonal central dogma. Nat Chem Biol 14, 103–106 (2018). https://doi.org/10.1038/nchembio.2554
  2. Ariel Hecht, Jeff Glasgow, Paul R. Jaschke, Lukmaan A. Bawazer, Matthew S. Munson, Jennifer R. Cochran, Drew Endy, Marc Salit, Measurements of translation initiation from all 64 codons in E. coli, Nucleic Acids Research, Volume 45, Issue 7, 20 April 2017, Pages 3615–3626, https://doi.org/10.1093/nar/gkx070
  3. Hutvagner, Andras Janos (2023). Developing a complete set of tRNAs to expand translation control. Macquarie University. Thesis. https://doi.org/10.25949/25039838.v1
  4. Scopelliti, Dominic (2024). Creating a library of initiator tRNA mutants to initiate protein translation from reprogrammed sense codons. Macquarie University. Thesis. https://doi.org/10.25949/28266281.v1
  5. Scopelliti, D., Vincent, R.M., Hutvagner, A., Whelan, F., Klare, W.P., Pang, I., Church, G.M., and Jaschke, P.R. (2025) Defining bottlenecks and physiological impact of an orthogonal translation initiation system. bioRxiv 2025.12.21.695813
  6. Scopelliti, D., Hutvagner, A., and Jaschke, P.R. (2026) Host background shapes the portability of a non-canonical translation initiation system across Escherichia coli strains. bioRxiv 2026.04.16.719103