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Query: EC:3.1.26.5 (RNase P)
 1,348 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.
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PMID:Characterization of ribonuclease P isolated from rat liver cytosol. 160 34

Processing of multimeric precursor tRNAs from Bacillus subtilis by the catalytic RNA component of RNase P was studied in vitro. Previous studies on processing by either Escherichia coli or B. subtilis RNase P-RNA utilized monomeric or dimeric substrates. In the experiments described here, a multimeric precursor tRNA containing six complete tRNA sequences and the partial sequence of a seventh were used. One species did not encode the 3'-terminal CCA sequence and the partial tRNA lacked 3' nucleotides and could form only a 3-base pair instead of a 7-base paired aminoacyl stem. Two species had the potential for forming extended base-paired aminoacyl stems. Processing was studied under varied ionic conditions. Chemical sequencing of the products showed that the RNase P-RNA cleavage produced the proper mature 5' termini for all of the six complete tRNA species, but no 5'-cleavage of the partial species was observed. At suboptimal ionic concentrations, the two species capable of forming extended base-paired aminoacyl stems were not observed. Thus, encoding of the 3'-CCA in a tRNA species is not critical for processing, but the formation of an aminoacyl stem with more than 3 base pairs is necessary. Particularly noteworthy was the observation that all species of the multimeric precursor could be processed at significantly lower ionic conditions than monomeric precursors used previously by ourselves and others. However, a single precursor species produced from the multimeric precursor could also be processed at the same lower ionic conditions as the multimeric precursor. This demonstrates that precursor tRNA species can differ widely in their ionic requirements for processing and that, to a large extent, the optimal conditions of MgCl2 or NH4Cl are a function of the substrate which is used.
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PMID:Processing of a multimeric tRNA precursor from Bacillus subtilis by the RNA component of RNase P. 313 56

The structure of M1 RNA, the RNA component of Escherichia coli RNase P, has been probed by mild digestion with a variety of ribonucleases. The results have been used to generate a model for the two-dimensional structure of M1 RNA. This model is similar in many respects to an earlier model that was based entirely on theoretical considerations. M1 RNA was digested with RNase T1 in buffer containing 10 mM MgCl2 (in which M1 RNA, by itself, has no catalytic activity) and in buffer containing 60 mM MgCl2 (in which M1 RNA can cleave precursors to tRNA molecules). Under these conditions, the main features of the secondary structure are similar, but several minor differences are apparent. Such subtle changes in structure are also observed when M1 RNA is present in a binary complex with a substrate molecule, the precursor to E. coli tRNATyr.
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PMID:Structure in solution of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli. 608 7

A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNA(Asp) catalyzed by the RNA component of B. subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris Cl (pH 8.0), 100 mM MgCl2, and 800 mM NH4Cl, 37 degrees C). This kinetic mechanism was derived from measuring pre-steady-state, steady-state, single-turnover, and binding kinetics using a combination of quench-flow, gel filtration, and gel shift techniques. A minimal kinetic description involves the following: (1) binding of pre-tRNA(Asp) to RNase P RNA rapidly (6.3 x 10(6) M-1 s-1), but slower than the diffusion-controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s-1; (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (> or = 0.2 s-1) followed by rate-limiting tRNA dissociation (0.02 s-1); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNA(Asp) significantly more slowly (7 x 10(4) M-1 s-1). This scheme involves the isolation of individual steps in the reaction sequence, is consistent with steady-state data, and pinpoints the rate-determining step under a variety of conditions. This kinetic mechanism will facilitate a more accurate definition of the role of metals, pH, and the protein component in each step of the reaction and provide an essential background for understanding the influence of structural changes on the catalytic activity.
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PMID:A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P. 752 Jul 53

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S. pombe RNase P, producing the mature 5' end of tRNA(Ser). pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl. The apparent Km for the S. pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.
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PMID:Partial purification and characterization of RNase P from Dictyostelium discoideum. 773 3

We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3' ends. The enzyme consists of one (or more) protein(s) without RNA subunits. Products of the processing reaction are tRNA molecules with 3' terminal hydroxyl groups and 3' trailers with 5' terminal phosphates. The main processing sites are located immediately 3' to the discriminator and one nucleotide further downstream. This endonucleolytic processing at and close to the tRNA 3' end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3' processing pathway. Partial purification and separation of RNase Z from the 5' processing activity RNase P allowed us to determine biochemical characteristics of the enzyme. The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30 degrees C. Optimal concentrations for MgCl2 and KCl are 5 mM and 30 mM, respectively. The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5' ends. The precursor for tRNAPhe requires RNA editing for efficient processing by RNase Z.
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PMID:5' end maturation and RNA editing have to precede tRNA 3' processing in plant mitochondria. 941 37

Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNAAsp cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including </=10-fold increases in the rate constant for tRNA dissociation, the affinity of tRNA, and the rate constant for phosphodiester bond cleavage. However, the protein principally affects substrate binding, increasing the affinity of RNase P for pre-tRNAAsp by a factor of 10(4) as determined from both the ratio of the pre-tRNAAsp dissociation and association rate constants measured in 10 mM MgCl2 and a binding isotherm measured in 10 mM CaCl2 using gel filtration to separate enzyme-bound and free pre-tRNAAsp. Therefore, the main role of the protein component in RNase P is to facilitate recognition of pre-tRNA by enhancing the interaction between the enzyme and the 5'-precursor segment of the substrate, rather than stabilizing the tertiary structure of the folded RNA as has been observed for protein-facilitated group I intron self-splicing. Furthermore, the protein component maximizes the efficiency of RNase P under physiological conditions and minimizes product inhibition.
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PMID:Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp. 948 87

Ribonuclease P (RNase P) contains a catalytic RNA that cleaves precursor tRNA (pre-tRNA) to form the mature 5'-end of tRNA. Previous kinetic analyses with mutant pre-tRNAs indicated that both C residues of the invariant 3'-terminal CCA form specific interactions with RNase P RNA that contribute to the energetics of substrate binding (1, 2). In the present study, we have used single-turnover kinetic analysis to investigate whether specific changes in the 3'-terminal CCA influence the rate of the chemical step through which enzyme-bound substrate is converted to product (k2). At optimal ionic strength (1.0 M NH4Cl, 25 mM MgCl2), deletion or substitution of the 3'-proximal C residue (CCA) reduced the rate of the chemical step of cleavage (k2) by 60-fold. Similar changes to the 5'-proximal C residue (CCA) or the 3'-terminal A residue (CCA) reduced k2 only a few fold. Each mutant substrate exhibited weakened affinity for Mg2+, as measured by Hill plots, and the severity of these defects correlated with the observed reductions in k2. Furthermore, elevated concentrations of Mg2+ partially, but not completely, suppress the k2 defects caused by deletion or substitution of the 3'-proximal C residue. We conclude that the 3'-CCA of pre-tRNA, particularly the 3'-proximal C residue, comprises part of the catalytic pocket formed in the pre-tRNA-RNase P complex and participates in the binding of Mg2+ ions that are essential for catalysis by RNase P RNA.
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PMID:Participation of the 3'-CCA of tRNA in the binding of catalytic Mg2+ ions by ribonuclease P. 958 41

Ribonuclease P (RNase P) is a key enzyme in tRNA biogenesis that catalyses the endonucleolytic cleavage of tRNA precursors and generates their mature 5' ends. The activity of this ribozyme has never been isolated from living human tissues and data about epidermal tRNA biogenesis are not available. The purpose of the present study was to isolate and purify RNase P from human epidermis and to investigate the in vitro effects of retinoids on its activity. Enzyme isolation and purification from homogenates of keratinocytes derived after trypsinization from dispase-separated human epidermis were carried out using phosphocellulose chromatography. The optimal activity of the enzyme was found at 100 mM NH4Cl and 5 mM MgCl2 at pH 7.5 and 37 degrees C. All-trans retinoic acid and acitretin revealed a dose-dependent inhibitory effect on RNase P activity. The isolation of RNase P activity from human epidermis, reported here for the first time, will enable the investigation of the possible involvement of this ribozyme in the regulation of epidermal differentiation and proliferation and the evaluation of its significance for the pathogenesis and gene therapy of various cutaneous disorders.
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PMID:Isolation of ribonuclease P activity from human epidermis and its regulation by retinoids in vitro. 1664 12