Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39.
Chlamydophila pneumoniae AR39 contains two different ORFs (CP0654 and CP0782) encoding ribonuclease H (RNase H) homologues, Cpn-RNase HII and Cpn-RNase HIII. Sequence alignments show that the two homologues both contain the conserved motifs of type 2 RNase H, and Cpn-RNase HII has the conserved active-site motif (DEDD) of RNase HII. Cpn-RNase HIII also contains a unique active-site motif (DEDE), common to other RNase HIIIs. Complementation assays indicated that Cpn-RNase HII can complement both Escherichia coli RNase HII and RNase HI, but Cpn-RNase HIII can only complement the latter. In vitro enzyme activity experiments showed that neither Cpn-RNase HII nor Cpn-RNase HIII is thermostable and their optimum pH values were 9.0 and 10.0, respectively. Cpn-RNase HII cleaves a 12 bp RNA-DNA substrate at multiple sites, but Cpn-RNase HIII at only one site. When a 35 bp DNA-RNA-DNA/DNA chimeric substrate was used, cleavage was only observed with Cpn-RNase HII. These results indicate that the RNase H combination of C. pneumoniae AR39 is not simple substitution of E. coli RNase H, perhaps representing a more primordial type. This is believed to be the first in vivo functional study of Chlamydophila RNase Hs and the results should contribute to the analysis of RNase Hs of other parasite species.
Liang
Rubing
R
College of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Liu
Xipeng
X
College of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Pei
Dongli
D
College of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Liu
Jianhua
J
College of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D016993:Chlamydophila pneumoniae / Q000201:enzymology*; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D012313:RNA; D004247:DNA; D012260:Ribonucleases; C091139:ribonuclease HII; C117079:ribonuclease HIII; D016914:Ribonuclease H; C078856:ribonuclease HI