title: GTP-dependent RNA 3'-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus.
abstract: We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Sato
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Soga
authors.forename: Kaori
authors.initials: K
authors.lastname: Igarashi
authors.forename: Kanako
authors.initials: K
authors.lastname: Takesue
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.forename: Akio
authors.initials: A
authors.lastname: Kanai
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism*; D010957:Plasmids; D011118:Polynucleotide Ligases / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial
chemical_list: D010710:Phosphates; D011994:Recombinant Proteins; D012313:RNA; D006160:Guanosine Triphosphate; D008025:Ligases; C037451:RNA 3'-terminal phosphate cyclase; D011118:Polynucleotide Ligases