title: Mutation of the gene encoding the ribonuclease P RNA in the hyperthermophilic archaeon Thermococcus kodakarensis causes decreased growth rate and impaired processing of tRNA precursors.
abstract: Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional functions, however, remain poorly understood. To gain insight into the biological function of archaeal RNase Ps in vivo, we prepared archaeal mutants KUWΔP3, KUWΔP8, and KUWΔP16, in which the gene segments encoding stem-loops containing helices, respectively, P3, P8 and P16 in RNase P RNA (TkopRNA) of the hyperthermophilic archaeon Thermococcus kodakarensis were deleted. Phenotypic analysis showed that KUWΔP3 and KUWΔP16 grew slowly compared with wild-type T. kodakarensis KUW1, while KUWΔP8 displayed no difference from T. kodakarensis KUW1. RNase P isolated using an affinity-tag from KUWΔP3 had reduced pre-tRNA cleavage activity compared with that from T. kodakarensis KUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUWΔP3 showed greater accumulation of unprocessed transcripts for pre-tRNAs than that of T. kodakarensis KUW1. The current study represents the first attempt to prepare mutant T. kodakarensis with impaired RNase P for functional investigation. Comparative whole-transcriptome analysis of T. kodakarensis KUW1 and KUWΔP3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps.
authors.affiliation: Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Ueda
authors.affiliation: Protein Chemistry and Engineering, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Ishino
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Suematsu
authors.affiliation: Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakashima
authors.affiliation: Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Kakuta
authors.affiliation: Laboratory of Functional Genomics of Extreamophiles, Graduate School, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kawarabayasi
authors.affiliation: Protein Chemistry and Engineering, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
authors.affiliation: Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. Electronic address: mkimura@agr.kyushu-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
mesh_terms: D049109:Cell Proliferation / Q000235:genetics*; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D043262:Ribonuclease P / Q000235:genetics*; D013329:Structure-Activity Relationship; D019712:Thermococcus / Q000502:physiology*
chemical_list: D012343:RNA, Transfer; D043262:Ribonuclease P
keywords: Hyperthermophilic archaea; Pre-tRNA processing; Ribonuclease P; Thermococcus kodakarensis