RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA–DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA–DNA hybrids than for RNA–RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA–RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β-sheet and the fifth α-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII–Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.
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Research Article|
February 24 2010
Characterization of RNase HII substrate recognition using RNase HII–argonaute chimaeric enzymes from Pyrococcus furiosus
Sayaka Kitamura;
Sayaka Kitamura
*Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
†Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan
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Kosuke Fujishima;
Kosuke Fujishima
*Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
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Asako Sato;
Asako Sato
*Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
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Daisuke Tsuchiya;
Daisuke Tsuchiya
*Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
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Masaru Tomita;
Masaru Tomita
*Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
†Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan
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Akio Kanai
Akio Kanai
1
*Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
†Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan
1To whom correspondence should be addressed (email akio@sfc.keio.ac.jp).
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Publisher: Portland Press Ltd
Received:
October 08 2009
Revision Received:
January 04 2010
Accepted:
January 05 2010
Accepted Manuscript online:
January 05 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 426 (3): 337–344.
Article history
Received:
October 08 2009
Revision Received:
January 04 2010
Accepted:
January 05 2010
Accepted Manuscript online:
January 05 2010
Citation
Sayaka Kitamura, Kosuke Fujishima, Asako Sato, Daisuke Tsuchiya, Masaru Tomita, Akio Kanai; Characterization of RNase HII substrate recognition using RNase HII–argonaute chimaeric enzymes from Pyrococcus furiosus. Biochem J 15 March 2010; 426 (3): 337–344. doi: https://doi.org/10.1042/BJ20091553
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