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We discovered that thePF1549gene inPyrococcus furiosusencodes a very heat-stable RNA 3′-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found thatPf-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 forPf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important forPf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity ofPf-Rtc is totally dependent on the linear form of the 3′-terminal phosphate, with an apparentKd value of 20 nm at 95 °C. Furthermore, we found thatPf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2′-5′ RNA ligase-like protein inP. furiosus). The possible roles ofPf-Rtc and the importance of terminal phosphate structures in RNA are discussed.