Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Li Tao Guo, Yane Shih Wang, Akiyoshi Nakamura, Daniel Eiler, Jennifer M. Kavran, Margaret Wong, Laura L. Kiessling, Thomas A. Steitz, Patrick O'Donoghue, Dieter Söll

Research output: Contribution to journalArticlepeer-review

62 Scopus citations


Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here,we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Original languageEnglish (US)
Pages (from-to)16724-16729
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number47
StatePublished - Nov 25 2014
Externally publishedYes


  • Aminoacyl-tRNA synthetase
  • Genetic code
  • Genetic selection
  • Posttranslational modification
  • Synthetic biology

ASJC Scopus subject areas

  • General


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