title: Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at TψC loop in precursor tRNAs.
abstract: Proteinaceous RNase P (PRORP1) in Arabidopsis thaliana is an endoribonuclease that catalyzes hydrolysis to remove the 5'-leader sequence of precursor tRNAs (pre-tRNAs). PRORP1 is composed of pentatricopeptide repeat (PPR) motifs, a central linker region, and a metal nuclease domain, the NYN domain. The PPR motifs are single-stranded RNA-binding motifs that recognize bases in a modular fashion. To obtain insight into the mechanism by which the PPR motifs in PRORP1 recognize a target sequence in catalysis, N-terminal successive deletion mutants were overproduced in Escherichia coli, and the resulting proteins were characterized in terms of enzymatic activity using chloroplast pre-tRNA(Phe) as a substrate. Although Δ89, in which all PPR motifs are present, retained the pre-tRNA cleavage activity, Δ129 devoid of the first PPR motif (PPR1) had significantly reduced cleavage activity. Likewise, deletions of the second (PPR2) or third PPR (PPR3) motif abolished the cleavage activity, suggesting that PPR motifs play a crucial role in catalysis. A proposed recognition code for PPR motifs predicted that PPR2-PPR5 in PRORP1 recognize C, A/U, A, and U, respectively, whose sequence is in good agreement with C56-A57-A58-A59 in the TψC loop in pre-tRNA(Phe). Mutational analyses of nucleotide residues in the TψC loop as well as nucleotide-specifying residues (NSRs) in PPR motifs further suggested that PPR2 and PPR3 in PRORP1 favorably recognize nucleotide bases C56 and A57 at the TψC loop in pre-tRNA(Phe), respectively. This prediction and previous biochemical data were combined to construct a fitting model of tRNA onto PRORP1, showing that the mechanism by which PRORP1 recognizes pre-tRNAs appears to be distinct from that by bacterial RNase P.
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Imai
authors.affiliation: Laboratory of Plant Biosciences, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Nakayama
authors.affiliation: Laboratory of Structural Biology, Graduate School of Systems Life Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Xuzhu
authors.initials: X
authors.lastname: Gao
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; Laboratory of Structural Biology, Graduate School of Systems Life Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakashima
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; Laboratory of Structural Biology, Graduate School of Systems Life Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Kakuta
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan; Laboratory of Structural Biology, Graduate School of Systems Life Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. Electronic address: mkimura@agr.kyushu-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D043262:Ribonuclease P / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D012343:RNA, Transfer; C581753:PRORP1 protein, Arabidopsis; D043262:Ribonuclease P
keywords: Arabidopsis thaliana; PRORP1; Pentatricopeptide repeat; tRNA precursor