Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

Akio Kanai, Asako Sato, Jun Imoto, Masaru Tomita

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75°C) with an apparent dissociation constant of 0.6 μM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

Original languageEnglish
Pages (from-to)373-379
Number of pages7
JournalBiochemical Journal
Issue number1
Publication statusPublished - 2006 Jan 1


  • Archaea
  • In vitro translation
  • Pyrococcus furiosus
  • RNA-binding protein
  • Stem-loop structure
  • Thymidylate synthase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein'. Together they form a unique fingerprint.

Cite this