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Purification and enzymatic characterization of the RNA ligase RTCB from Thermus thermophilus

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Abstract

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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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFA0500600, 2015CB943300), the National Natural Science Foundation of China (31670878) and the Shanghai Committee of Science and Technology (18430711400) to J.L.

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Collaborative Innovation Center of Genetics and Development, Fudan University, 200438, Shanghai, China

    Shuyan Duan, Zijun Chen, Zhengyang Li, Rui Ji, Jianhua Gan & Jixi Li

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  1. Shuyan Duan
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  2. Zijun Chen
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  3. Zhengyang Li
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  4. Rui Ji
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  5. Jianhua Gan
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  6. Jixi Li
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Correspondence to Jixi Li.

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Duan, S., Chen, Z., Li, Z. et al. Purification and enzymatic characterization of the RNA ligase RTCB from Thermus thermophilus. Biotechnol Lett 41, 1051–1057 (2019). https://doi.org/10.1007/s10529-019-02707-0

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