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

Toshifumi Ueda, Sonoko Ishino, Kotaro Suematsu, Takashi Nakashima, Yoshimitsu Kakuta, Yutaka Kawarabayasi, Yoshizumi Ishino, Makoto Kimura

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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.

Original languageEnglish
Pages (from-to)660-665
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume468
Issue number4
DOIs
Publication statusPublished - Dec 25 2015

Fingerprint

Thermococcus
Ribonuclease P
Gene encoding
Archaea
RNA Precursors
RNA
Ribonucleases
Mutation
Growth
Processing
Genes
Archaeal RNA
Gene Expression Profiling
Eukaryotic Cells
Northern Blotting
Polymerase Chain Reaction
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

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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.",
author = "Toshifumi Ueda and Sonoko Ishino and Kotaro Suematsu and Takashi Nakashima and Yoshimitsu Kakuta and Yutaka Kawarabayasi and Yoshizumi Ishino and Makoto Kimura",
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T1 - 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

AU - Ueda, Toshifumi

AU - Ishino, Sonoko

AU - Suematsu, Kotaro

AU - Nakashima, Takashi

AU - Kakuta, Yoshimitsu

AU - Kawarabayasi, Yutaka

AU - Ishino, Yoshizumi

AU - Kimura, Makoto

PY - 2015/12/25

Y1 - 2015/12/25

N2 - 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.

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