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Updated: Jan 22, 2026

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
Purification and enzymatic characterization of the RNA ligase RTCB from Thermus thermophilus
Shuyan Duan1, Zijun Chen1, Zhengyang Li1
1State Key Laboratory of Genetic Engineering, School of Life Sciences, Collaborative Innovation Center of Genetics and Development, Fudan University, 200438, Shanghai, China.
Objective:
To identify the key residues of Thermus thermophilus (T. thermophilus) RTCB in RNA ligation and DNA activation.
Results:
The 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 °C.
Conclusion:
D64 was proved to be essential for RTCB-catalyzed RNA ligation and DNA activation (from 37 to 70 °C) in T. thermophilus.
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