Characterization of a new RNase HII and its essential amino acid residues in the archaeon Sulfolobus tokodaii reveals a regulatory C-terminus.
The archaea possess RNase H proteins that share features of both prokaryotic and eukaryotic forms. Although the Sulfolobus RNase HI has been reported to have unique structural and biochemical properties, its RNase HII has not yet been investigated and its biochemical properties remain unknown. In the present study, we have characterized the ST0519 RNase HII from S. tokodaii as a new form. The enzyme utilized hybrid RNA/DNA as a substrate and had an optimal temperature between 37 to 50 degrees C. The activity of wild-type protein was stimulated by Mn2+, whereas this cation significantly inhibited the activity of C-terminal truncated mutant proteins. A series of mutation assays revealed a regulatory C-terminal tail in the S. tokodaii RNase HII. One mutant, ST0519 (residues 1-195), retained only partial activity, while ST0519 (residues 1-196) completely lost its activity. Based on the presumed structure, the C-terminus might form a short alpha-helix in which two residues, I195 and L196, are essential for the cleavage activity. Our data suggest that the C-terminal alpha-helix is likely involved in the Mn2+-dependent substrate cleavage activity through stabilization of a flexible loop structure. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNase HII.
Zhan
Ke
K
Center for Proteomics Research, Huazhong Agricultural University, Wuhan, China.
He
Zheng-Guo
ZG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; C091139:ribonuclease HII; D016914:Ribonuclease H