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 KUW Delta P3, KUW Delta P8, and KUW Delta P16, 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 KUW Delta P3 and KUW Delta P16 grew slowly compared with wild-type T. kodakarensis KUW1, while KUW Delta P8 displayed no difference from T kodakarensis KUW1. RNase P isolated using an affinity-tag from 1(KUW Delta P3 had reduced pre-tRNA cleavage activity compared with that from T kodakarensis KUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUW Delta P3 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 KUW Delta P3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps. (C) 2015 Elsevier Inc. All rights reserved.