title: Mutational analysis of the length of the J3/4 domain of Escherichia coli ribonuclease P ribozyme.
abstract: We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A(62)G(63)G(64)A(65)) was replaced with GGA (denoted DeltaA), GA (DeltaAG), A (DeltaAGG), AAGGA (SigmaA), AAAGGA (SigmaAA), and AAAAGGA (SigmaAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>>SigmaAA>SigmaA>SigmaAAA>>DeltaAG>DeltaA, DeltaAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.
authors.affiliation: Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempakkucho, Toyohashi, Aichi 441-8580, Japan.
authors.forename: Shinnosuke
authors.initials: S
authors.lastname: Haga
authors.forename: Terumichi
authors.initials: T
authors.lastname: Tanaka
authors.forename: Yo
authors.initials: Y
authors.lastname: Kikuchi
mesh_terms: D002384:Catalysis; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016337:RNA, Catalytic / Q000235:genetics*; D043262:Ribonuclease P
chemical_list: D016337:RNA, Catalytic; D043262:Ribonuclease P; D008274:Magnesium