Functional Characterization Of An Archaeal RNase HⅡ

RNase H is a class of nucleic acid hydrolase which widely exist in biosystem. They participate in controlling the type and quantity of intracellular RNA, involving the process such as cleavage,modification and degradation. Archaea are the third domain of life, and the function and the mechanism of their RNase H remain unkown. In this study, we choose RNase HⅡof Sulfolobus tokodaii strain 7 as a model and analyzed their activity under different conditions. Through the design of site-specific mutants and deletion mutants, we identified the critical amino acids which determine the activity of the protein, as well as the possible structural mechanism. Results are as follows:(1) The function of RNase HⅡ(ST0519) of S. tokodaii strain 7 was identified and the best reaction conditions for wild-type ST0519 protein, such as temperature, pH value, and metal ions, were figured out. (2) The site-specific mutants were designed and their proteins were purified. Further activities discovered several critical amino acid sites, including Asp7, Glu8, Ile195, and Leu196, which significantly affect the activity of ST0519. (3) The deletion mutants were produced and their proteins were successfully purified. Theα-helix in the C-terminus was unexpectedly found to have a major impact on the activity of ST0519. (4) Metal ions stimulated the activity of wild-type protein, but inhibited the activity of mutant protein.It is the first report on the identification of the function and regulation mechanism of S. tokodaii strain 7 RNase HⅡ. On the one hand, our results demonstrated that the archaeal ST0519 protein had a similar characteristic to the other previously reported RNase H. On the other hand, a C-terminalα-helix of the archaeal protein was found to play an important role, indicating that it would be a new regulatory mechanism in the archaeon. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNaseHⅡ.