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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)

A high yield, photoactivated cross-linking reaction between a modified tRNA and RNase P RNA was used as a quantitative assay of substrate binding affinity. The cross-linking assay allows the effects of metal ions on substrate binding to be measured independently and in the absence of the pre-tRNA cleavage reaction. The results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent cations act primarily to enhance enzyme-substrate binding, presumably by functioning as counterions. This enhancement can be attributed to a reduction in the tRNA off-rate. (ii) Although divalent cation is required for cleavage, the enzyme-substrate complex can form in the absence of divalent cation; the essential role of divalent cation in the reaction is thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but supports catalysis only at a low rate.
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PMID:Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis. 137 Aug 19

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.
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PMID:Purification and characterization of RNase P from Clostridium sporogenes. 170 96

The location of phosphate residues involved in specific centers for binding of metal ions in M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, was determined by analysis of induction of cleavage of RNA by metal ions. At pH 9.5, Mg2+ catalyzes cleavage of M1 RNA at five principal sites. Under certain conditions, Mn2+ and Ca2+ can each replace Mg2+ as the cofactor in the processing of precursor tRNAs by M1 RNA and P RNA, the RNA subunit of RNase P from Bacillus subtilis. These cations, as well as various metal ion inhibitors of the catalytic activity of M1 RNA, also promote cleavage of M1 RNA in a specific manner. Certain conditions that affect the catalytic activity of M1 RNA also alter the rate of metal ion-induced cleavage at the various sites. From these results and a comparison of cleavage of M1 RNA with that of a deletion mutant of M1 RNA and of P RNA, we have identified two different centers for binding of metal ions in M1 RNA that are important for the processing of the precursor to tRNA(Tyr) from E. coli. There is also a center for the binding of metal ions in the substrate, close to the site of cleavage by M1 RNA.
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PMID:Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. 171

A synthetic tRNA precursor analog containing the structural elements of Escherichia coli tRNA(Phe) was characterized as a substrate for E. coli ribonuclease P and for M1 RNA, the catalytic RNA subunit. Processing of the synthetic precursor exhibited a Mg2+ dependence quite similar to that of natural tRNA precursors such as E. coli tRNA(Tyr) precursor. It was found that Sr2+, Ca2+, and Ba2+ ions promoted processing of the dimeric precursor at Mg2+ concentrations otherwise insufficient to support processing; very similar behavior was noted for E. coli tRNA(Tyr). As noted previously for natural tRNA precursors, the absence of the 3'-terminal CA sequence in the synthetic precursor diminished the facility of processing of this substrate by RNase P and M1 RNA. A study of the Mg2+ dependence of processing of the synthetic tRNA dimeric substrate radiolabeled between C75 and A76 provided unequivocal evidence for an alteration in the actual site of processing by E. coli RNase P as a function of Mg2+ concentration. This property was subsequently demonstrated to obtain (Carter, B. J., Vold, B.S., and Hecht, S. M. (1990) J. Biol. Chem. 265, 7100-7103) for a mutant Bacillus subtilis tRNAHis precursor containing a potential A-C base pair at the end of the acceptor stem.
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PMID:Metal ion and substrate structure dependence of the processing of tRNA precursors by RNase P and M1 RNA. 226 41

We have studied cleavage site selection by M1 RNA, the catalytic subunit of Escherichia coli RNase P, under various reaction conditions using tRNA precursors which are cleaved at two positions. Our results showed that the preference of cleavage site changed with variations in pH or Mg2+ concentration. By contrast, no difference in cleavage site selection was observed with increasing pH in the presence of Ca2+ as the only divalent metal ion. Depending on the identity of the nucleotide at position "+ 72", replacement of Mg2+ with Ca2+ resulted in a change of the main cleavage site irrespective of pH. We conclude that cleavage in the presence of Ca2+ compared to cleavage in the presence of Mg2+ has different structural requirements at and near the cleavage site. UV cross-linking revealed that close points between M1 RNA and its substrate were the same irrespective of pH or the identity of the divalent cation. Our results also showed that the observed pH effects are due to changes in the catalytic cleavage rates rather than to global, structural rearrangements. These data are discussed in terms of metal ion binding near the cleavage sites in the enzyme-substrate complex.
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PMID:Cleavage site selection by M1 RNA the catalytic subunit of Escherichia coli RNase P, is influenced by pH. 752 66

We synthesized a chimeric RNA between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a model substrate of the enzyme. The model substrate is the smallest substrate of RNase P, having a simple stem-loop structure. This model substrate was added to the 3'-end of M1 RNA. This chimeric molecule, which we call M1 RNA-MS, is a self-cleaving RNA and is cleaved much more efficiently than the M1 RNA-pre-tRNA, an artificial self-cleaving RNA previously synthesized [Kikuchi et al. (1993) Nucleic Acids Res. 21, 4685-4689], that consists of a full-size tRNA precursor and the M1 RNA. The self-cleavage of M1 RNA-MS at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage). Ca2+ supported the self-cleavage of M1 RNA-MS as effectively as Mg2+, although the self-cleavage of M1 RNA-pre-tRNA proceeded with low efficiency in the presence of Ca2+ as the only metal ion. Future application of the M1 RNA-MS molecule to the in vitro evolution of the M1 RNA and other experiments is proposed.
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PMID:Synthesis and self-cleavage reaction of a chimeric molecule between RNase P-RNA and its model substrate. 753 87

A gel retardation assay has been used to examine the kinetic and equilibrium properties of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli. The interaction is relatively insensitive to the identity of the monovalent anions present and to pH in the range 7.0-9.0, but it has a more critical requirement for specific monovalent and divalent cations: NH4+, K+, Mg2+, Ca2+, and Mn2+ all promote efficient formation of the complex. A positive delta S (+6.4 cal mol-1 deg-1) and a negative delta H (-11.3 kcal mol-1) combine to give a delta G equal to -13.3 kcal mol-1 at 37 degrees C in 0.42 M salt. The binding reaction is sensitive to the concentration of monovalent and divalent cations, with the affinity increasing with increasing ionic strength (delta log Ka/delta log [NH4+] = +2.7 +/- 0.1). The dependence of Kd on the ionic strength and the positive delta S suggests that hydrophobic and stacking interactions contribute significantly to the formation of the RNase P holoenzyme.
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PMID:Kinetic and thermodynamic analysis of RNA-protein interactions in the RNase P holoenzyme from Escherichia coli. 831 59

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.
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PMID:In vitro selection for altered divalent metal specificity in the RNase P RNA. 940 16

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.
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PMID:Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity. 979 10

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.
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PMID:Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA. 1099 99


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