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

• Published in: Biochemical and biophysical research communications Vol. 468; no. 4; pp. 660 - 665
• Main Authors: Ueda, Toshifumi, Ishino, Sonoko, Suematsu, Kotaro, Nakashima, Takashi, Kakuta, Yoshimitsu, Kawarabayasi, Yutaka, Ishino, Yoshizumi, Kimura, Makoto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.12.2015
• Subjects: Cell Proliferation - genetics; Enzyme Activation; Hyperthermophilic archaea; Mutagenesis, Site-Directed; Mutation - genetics; Pre-tRNA processing; Ribonuclease P; Ribonuclease P - genetics; RNA, Transfer - genetics; Structure-Activity Relationship; Thermococcus; Thermococcus - physiology; Thermococcus kodakarensis; Thermococcus kodakarensis; CBP; 5-FOA; Ribonuclease P; RNase P; Pre-tRNA processing; ASW; PhopRNA; qRT-PCR; Hyperthermophilic archaea; pre-tRNA; TkopRNA
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.11.012

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. •RNase P plays essential role in non-tRNA biogenesis in bacterial and eukaryotic cells.•The mutant cell grew slowly compared with wild-type Thermococcus kodakarensis.•RNase P isolated from the mutant cell had reduced pre-tRNA cleavage activity.•The mutant cell accumulated unprocessed transcripts for pre-tRNAs.•The result demonstrates the preparation of the archaeal mutant with impaired RNase P.