TAILIEUCHUNG - Báo cáo khoa học: Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium

The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacteriumShewanellasp. SIB1, over-produced inEscherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% toE. coliRNase HI, and 32% to human RNase H1. | ỊFEBS Journal Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium Takashi Tadokoro1 Hyongi Chon1 Yuichi Koga1 Kazufumi Takano1 2 and Shigenori Kanaya1 1 Department of Materialand Life Science Graduate Schoolof Engineering Osaka University Japan 2 CRESTO JST Osaka Japan Keywords double-stranded RNA binding domain RBD gel mobility shift assay psychrotrophic bacterium RNase H Shewanella sp. SIB1 Correspondence S. Kanaya Department of Materialand Life Science Graduate School of Engineering Osaka University 2-1 Yamadaoka Suita Osaka 565-0871 Japan Fax 81 6 6879 7938 Tel 81 6 6879 7938 E-mail kanaya@ Present address Laboratory of Molecular Genetics National Institute of Health Bethesda MD USA Received 16 February 2007 revised 28 May 2007 accepted 29 May 2007 doi The gene encoding a bacterial type 1 RNase H termed RBD-RNase HI was cloned from the psychrotrophic bacterium Shewanella sp. SIB1 overproduced in Escherichia coli and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26 to SIB1 RNase HI 17 to E. coli RNase HI and 32 to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain RBD at the N-terminus which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA DNA hybrid in an isolated form suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI E. coli RNase HI and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI .

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