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

Biotechnol Lett. 2019 Sep;41(8-9):1051-1057. doi: 10.1007/s10529-019-02707-0. Epub 2019 Jul 6.

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.

Keywords: DNA activation; Purification; RNA ligation; RTCB.

MeSH terms

  • Conserved Sequence
  • DNA / metabolism*
  • DNA Mutational Analysis
  • RNA / metabolism*
  • RNA Ligase (ATP) / genetics
  • RNA Ligase (ATP) / isolation & purification*
  • RNA Ligase (ATP) / metabolism*
  • Sequence Homology, Amino Acid
  • Temperature
  • Thermus thermophilus / enzymology*

Substances

  • RNA
  • DNA
  • RNA Ligase (ATP)