title: Characterization of the peripheral structures of archaeal RNase P RNA from Pyrococcus horikoshii OT3.
abstract: Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5' leader sequence of precursor tRNA (pre-tRNA). The secondary structure of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 has several peripheral stem-loops. In order to investigate their functional role, we prepared six mutants, ΔP1, ΔP3, ΔP8, ΔP9, ΔP12 and ΔP15, in which the stem-loops including helices P1, P3, P8, P9, P12/12.1/12.2 and P15/16 in PhopRNA were individually deleted, respectively, and characterized them with respect to pre-tRNA cleavage activity in the presence of five proteins and also to the ability to form a complex with the proteins. The reconstituted particles containing ΔP3, ΔP8 or ΔP9 retained considerable levels of activity (35-65%), while those containing ΔP1, ΔP12 or ΔP15 had markedly reduced activity (13%). It was further found that the reconstituted particles comprising ΔP3 or ΔP15 lacked PhoPop5 and PhoRpp30, whereas those containing ΔP1, ΔP8, ΔP9 or ΔP12 bound to all five proteins. Since it is known that PhoPop5 functions in a complex with PhoRpp30, the present result suggests that the peripheral stem-loops containing P3 or P15/16 are involved in the structural formation of a catalytic site by interacting with the protein complex PhoPop5-PhoRpp30.
authors.affiliation: Laboratory of Structural Biology, Graduate School of Sytems Life Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan; and Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Ueda
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Miyanoshita
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakashima
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Kakuta
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
mesh_terms: D001483:Base Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D019642:RNA, Archaeal / Q000737:chemistry* / Q000235:genetics; D043262:Ribonuclease P / Q000235:genetics*
chemical_list: D019642:RNA, Archaeal; D043262:Ribonuclease P
keywords: Pyrococcus horikoshii; archaea; ribonuclease P; ribozyme; tRNA