Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein.
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 degrees C) with an apparent dissociation constant of 0.6 microM. 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.
Kanai
Akio
A
Institute for Advanced Biosciences, Keio University, Tsuruoka, 997-0017, Japan. akio@sfc.keio.ac.jp
Sato
Asako
A
Imoto
Jun
J
Tomita
Masaru
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014176:Protein Biosynthesis; D020027:Pyrococcus furiosus / Q000201:enzymology*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013940:Thymidylate Synthase / Q000378:metabolism*
D016601:RNA-Binding Proteins; D013940:Thymidylate Synthase