ObjectiveTo identify the key residues of Thermus thermophilus (T. thermophilus) RTCB in RNA ligation and DNA activation.ResultsThe biochemical activities of RTCB from T. thermophilus were purified, characterized, and compared. Structure and sequence alignment between T. thermophilus RTCB and Pyrococcus horikoshii (P. horikoshii) RTCB identified six conserved residues (D64, D95, N203, H204, E207, H399) that were essential for RNA ligation. Mutation analysis showed that the expression levels of mutants D95A, N203A, H204A, E207A and H399A were relatively low. Compared to wide-type RTCB, variant D64A protein had no RNA ligation and DNA activation activity. In addition, T. thermophilus RTCB showed acceptable catalytic activity of 3-phosphate DNA activation at 37 degrees C.Conclusion D64 was proved to be essential for RTCB-catalyzed RNA ligation and DNA activation (from 37 to 70 degrees C) in T. thermophilus.