Characterization and partial purification of two pre‐tRNA 5′‐processing activities from Daucus carrota (carrot) suspension cells

Abstract

SummaryTwo distinct RNase P-like activities which cleave leader sequences from pre-tRNA molecules to give mature 5' ends have been identified in carrot suspension-culture cells. An Escherichia coil pre-tRNA Pbe and a tobacco pretRNA Tyr were transcribed in vitro then used as substrates for processing reactions in a call-free extract. The pretRNA Tyr transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5' and 3' processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5' ends of both pre-tRNAs. Subsequent fractionation of the 5' end-procassing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCl, when assayed with the tobacco pretRNA Tyr substrate. When the same fractions were assayed with the E. coil pre-tRNA Phe, only the 0.1 M KCl fraction exhibited activity. Both of the active fractions display sensitivity to micrococcal nucleasa (MN) and proteinasa K indicating each is a ribonucleoprotein, a result not seen with other plant RNasa Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

“…We have used some of these naturally occurring mutants to determine the influence of the implied structural alterations in the corresponding pre-tRNAs on cleavage by purified wheat nuclear RNase P. Tn contrast to the Escherichiu coli enzyme, which recognizes a variety of substrates diverging from the canonical tRNA structure Guerrier-Takada et al, 1988;Forster and Altman, 1990), the wheat enzyme is very sensitive against structural aberrations: changes involving the T-stem and T-loop domain, and even the presence of an intron in the anticodon loop, significantly reduce the cleavage efficiency. The disruption of a tertiary interaction between T-loop and D-loop in NtS4 (this study), like the interruption of the anticodon stem in AtSS , leads to complete loss of processing by wheat germ RNase P. This sensitivity against deviations from the conserved pre-tRNA structure is consistent with the observation made for RNase P from carrot cells, which does not cleave a minimal substrate devoid of the D-loop domain (Franklin et al, 1995). In contrast, the enzymes from HeLa (Yuan and Altman, 1994, 199.5) and Xenopus nuclei (Carrara et al, 1995) tolerate alterations of the substrate structure, including disruption or deletion of D-stem and anticodon-stem domains, as long as the acceptorstem and T-loop remain unchanged.…”

5′ end maturation and RNA editing have to precede tRNA 3′ processing in plant mitochondria

“…5Ј processing catalyzed by RNase P thus has to precede 3Ј processing by RNase Z in a potato mitochondrial extract. In this respect the potato mitochondrial RNase Z resembles most of the tRNA 3Ј endonucleases characterized in other organisms (5,7,8,11,(35)(36)(37)(38), including carrot nuclei (9). The opposite processing order (3Ј processing preceding 5Ј processing) has been reported for Bombyx mori (10) and wheat germ nuclear͞ cytoplasmic extracts (39), whereas in pea and spinach chloroplast (40), as well as in wheat mitochondria (4), 5Ј and 3Ј processing activities have been found to be independent of each other.…”

Mitochondrial RNase P RNAs in ascomycete fungi: Lineage-specific variations in RNA secondary structure

“…A secondary structure model of the eukaryotic P-RNA conforms convincingly to the bacterial consensus structure with only minor deviations (Chen and Pace 1997;Frank et al 2000). In plants, RNase P has been purified, and a P-RNA component has been suggested based on the sensitivity of the carrot RNase P activity to micrococcal nuclease treatment (Franklin et al 1995). Although the nuclear RNase P from wheat is resistant to nuclease treatment, its density and its low isoelectric point also indicate the presence of a P-RNA subunit (Arends and Schön 1997).…”

“…Mitochondrial RNase P activities have been studied in various yeasts (see below), the ascomycete fungus Aspergillus nidulans (Lee et al 1996a), human (Doersen et al 1985;Rossmanith and Karwan 1998;Puranam and Attardi 2001;Rossmanith and Potuschak 2001), Trypanosoma brucei (Salavati et al 2001), potato (Marchfelder and Brennicke 1994), wheat (Hanic-Joyce and Gray 1990), and carrot (Franklin et al 1995). The most detailed information on the biochemical and genetic properties of mitochondrial RNase P (mtP-RNA) is available for Saccharomyces cerevisiae.…”