Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon.
We have cloned the gene encoding RNase HII (RNase HIIPk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli. This gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII. RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity was determined at 30 degreesC and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E. coli RNase HII, and Mg2+ for E. coli RNase HI. The specific activity of RNase HIIPk determined in the presence of the most preferred metal ion was 6. 8-fold higher than that of E. coli RNase HII and 4.5-fold lower than that of E. coli RNase HI. Like E. coli RNase HI, RNase HIIPk and E. coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3' bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HIIPk and E. coli RNases HI and HII are structurally and functionally related to one another.
Haruki
M
M
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Hayashi
K
K
Kochi
T
T
Muroya
A
A
Koga
Y
Y
Morikawa
M
M
Imanaka
T
T
Kanaya
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019766:Genes, Archaeal; D008969:Molecular Sequence Data; D009154:Mutation; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics*; D019642:RNA, Archaeal / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D019641:DNA, Archaeal; D019642:RNA, Archaeal; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; C091139:ribonuclease HII; D016914:Ribonuclease H