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Related Experiment Video

Updated: Aug 27, 2025

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli
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Ancestral archaea expanded the genetic code with pyrrolysine.

Li-Tao Guo1, Kazuaki Amikura2, Han-Kai Jiang3

  • 1Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, USA.

The Journal of Biological Chemistry
|September 24, 2022
PubMed
Summary

Pyrrolysyl-tRNA synthetase (PylRS) enzymes lacking a key domain remain active and orthogonal. This study characterizes a novel PylRS, revealing an expanded substrate spectrum and ancient origins of the PylRS-tRNA pair.

Keywords:
amber suppressionaminoacyl-tRNA synthetasegenetic code expansionnoncanonical amino acidspyrrolysyl-tRNA synthetasesynthetic biology

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Evolutionary Biology

Background:

  • Pyrrolysyl-tRNA synthetase (PylRS) is crucial for incorporating the 22nd amino acid, pyrrolysine, into proteins.
  • PylRS orthogonality and substrate tolerance make it a key tool for genetic code expansion.
  • A novel class of PylRS enzymes (ΔPylSn) lacking the N-terminal tRNA-binding domain has been identified in methanogenic archaea.

Purpose of the Study:

  • To biochemically and functionally characterize a ΔPylSn class PylRS from Candidatus Methanomethylophilus alvus.
  • To investigate the evolutionary history and coevolution of PylRS and pyrrolysine tRNA (tRNAPyl).
  • To explore the potential for engineering PylRS systems for expanded genetic codes.

Main Methods:

  • Biochemical assays to determine aminoacylation efficiency.
  • In vivo UAG-readthrough assays to assess protein synthesis.
  • Molecular phylogenetic analysis of PylRS and tRNAPyl sequences.

Main Results:

  • The Ca. M. alvus ΔPylSn PylRS exhibits reduced aminoacylation efficiency but an expanded amino acid substrate spectrum compared to full-length PylRS.
  • Phylogenetic analysis indicates the PylRS•tRNAPyl pair diverged early in evolutionary history, predating the three domains of life.
  • Emergence of tRNAPyl variants with unique discriminator bases (A73, U73) was observed, alongside coevolutionary patterns with PylRS.

Conclusions:

  • ΔPylSn PylRS enzymes represent a distinct class with altered substrate specificity and maintained orthogonality.
  • The ancient divergence of the PylRS•tRNAPyl pair suggests a long evolutionary history for pyrrolysine incorporation.
  • Engineered PylRS systems utilizing novel tRNAPyl variants hold promise for advancing genetic code expansion technologies.