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.
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.
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超嗜热古菌 Thermococcus kodakarensis 中编码核糖核酸酶 P RNA 的基因突变会导致生长速度下降和 tRNA 前体加工受损。
DOI：
10.1016/j.bbrc.2015.11.012
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
M.
中科院分区：
文献类型：
--
作者：
Ueda;T.;Ishino;S.;Suematsu K.;Nakashima;T.;Kakuta;Y.;Kawarabayasi;Y.;Ishino;Y.;and Kimura;M.
核糖核酸酶P（RNase P）催化所有三个系统发育域中tRNA前体的5′前导序列的加工。RNase P也在细菌和真核细胞中的非tRNA生物合成中起重要作用。然而，对于古细菌RNase Ps，其他功能仍然知之甚少。为了深入了解古细菌RNase Psin的生物学功能，我们制备了古细菌突变体KUWΔP3、KUWΔP8和KUWΔP16，它们分别缺失了超嗜热古细菌Thermococcus kodakarensis RNase PRNA（TkopRNA）中编码含螺旋茎环的基因片段P3、P8和P16。表型分析表明，KUWΔP3和KUWΔP16与野生型柯氏锥虫KUW 1相比生长缓慢，而KUWΔP8与野生型柯氏锥虫KUW 1相比生长无明显差异。使用亲和标签从KUWΔP3分离的RNase P与来自T. kodakarensisKUW 1的相比具有降低的前tRNA切割活性。此外，KUWΔP3的定量RT-PCR（qRT-PCR）和北方印迹分析显示，与柯达嗜热厌氧菌KUW 1相比，前体tRNA的未加工转录物的积累更大。本研究首次尝试制备RNase P受损的mucobacterium kodakarensis用于功能研究。对KodakarensisKUW 1和KUWΔP3的比较全转录组分析应该允许全面鉴定古细菌RNase Ps的RNA底物。
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 Psin 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 archaeonThermococcus kodakarensiswere deleted. Phenotypic analysis showed that KUWΔP3 and KUWΔP16 grew slowly compared with wild-typeT. kodakarensisKUW1, while KUWΔP8 displayed no difference fromT. kodakarensisKUW1. RNase P isolated using an affinity-tag from KUWΔP3 had reduced pre-tRNA cleavage activity compared with that fromT. kodakarensisKUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUWΔP3 showed greater accumulation of unprocessed transcripts for pre-tRNAs than that ofT. kodakarensisKUW1. The current study represents the first attempt to prepare mutantT. kodakarensiswith impaired RNase P for functional investigation. Comparative whole-transcriptome analysis ofT. kodakarensisKUW1 and KUWΔP3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps.