Ribonuclease H (RNase H) endonucleolytically hydrolyzes the RNA of RNA/DNA hybrids in the presence of divalent cations (Crouch & Dirksen, 1982
). The enzyme is widely present in various organisms. The RNase H enzymes are classified into two major families, type 1 and type 2, based on amino-acid sequence differences (Ohtani et al.
). These enzymes are thought to be involved in DNA replication, repair and/or transcription (Kogoma & Foster, 1998
; Murante et al.
; Qiu et al.
; Rydberg & Game, 2002
In prokaryotes, three different RNases H (RNases HI, HII and HIII) have so far been identified (Ohtani et al.
; Itaya et al.
). RNases HI are members of the type 1 RNase H family, while RNases HII and HIII are members of the type 2 RNase H family. Crystal structures are available for RNases HI (Katayanagi et al.
; Yang et al.
; Ishikawa et al.
) and HII (Lai et al.
; Muroya et al.
; Chapados et al.
). Comparison of these structures indicates that RNases HI and HII share a main-chain fold containing four acidic active-site residues, despite the lack of any significant amino-acid sequence similarity. For Escherichia coli
RNase HI, a catalytic mechanism and a substrate-recognition mechanism have been proposed (Kanaya, 1998
; Goedken & Marqusee, 2001
; Tsunaka et al.
). However, compared with RNases HI and HII, much less is known about the structure and function of RNase HIII as no structural information is available.
RNase HIII shows poor amino-acid sequence identity to RNase HII (Ohtani et al.
). For example, Bacillus subtilis
RNase HIII shows 18% amino-acid sequence identity to B. subtilis
RNase HII. Nevertheless, they are classified into the same family because several sequence motifs are conserved in their sequences. Single bacterial cells usually contain two different RNases H (Ohtani et al.
). For example, E. coli
cells contain RNases HI and HII, while B. subtilis
cells contain RNases HII and HIII. Biochemical characterization of B. subtilis
RNase HIII indicated that this enzyme is more closely related to RNase HI than to RNase HII in enzymatic properties, suggesting that RNase HIII functions as a substitute for RNase HI in B. subtilis
(Ohtani et al.
We have recently cloned the gene encoding RNase HIII from the thermophilic bacterium B. stearothermophilus
(Bst RNase HIII) and biochemically characterized the recombinant protein (Chon et al.
). Bst RNase HIII is composed of 310 amino-acid residues and shows an amino-acid sequence identity of 47.1% to B. subtilis
RNase HIII. Bst RNase HIII closely resembles B. subtilis
RNase HIII in enzymatic properties, such as its requirement for divalent cations, its pH optimum and the cleavage mode of the substrate. However, Bst RNase HIII is much more stable than B. subtilis
RNase HIII, suggesting that Bst RNase HIII is more suitable for crystallographic studies than B. subtilis
RNase HIII. Crystallization and preliminary X-ray crystallographic analysis of B. subtilis
RNase HIII have been reported (Kwak et al.
); however, this crystal structure has not yet been solved.
Here, we report the crystallization and preliminary crystallographic studies of Bst RNase HIII.