Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Ribonuclease P (RNase P) is the enzyme responsible for endonucleolytically separating the 5'-leader sequence from precursor tRNA molecules. In bacteria, and in the nuclei and mitochondria of all eukaryotes studied so far, RNase P contains an RNA subunit which is necessary for activity in vitro and in vivo. In contrast, we showed earlier that partially-purified RNase P from spinach chloroplasts had physical properties inconsistent with the presence of any RNA. We now report that the properties of the chloroplast enzyme, after 500 to 1500-fold purification, are consistent with enzymatic activity residing in a approximately 70 kDa polypeptide. Gel filtration chromatography on Sephacryl S-200 and S-300 provides a mass for chloroplast RNase P of approximately 70 +/- 5 kDa. A single polypeptide of approximately 70-80 kDa can be crosslinked to iodoUMP-substituted pre-tRNA. The labeling intensity of this polypeptide corresponds closely to the peak of RNase P activity on Sephacryl S-200 chromatography. Unlike the bacterial ribozyme-type RNase P, chloroplast RNase P is not a metalloenzyme. We showed previously that phosphodiester bond cleavage by the E. coli RNA enzyme absolutely requires Mg2+ or Mn2+ coordinated to the pro-Rp oxygen of the scissile phosphodiester phosphate. In contrast, we now find that chloroplast RNase P has no such requirement, and can accurately and efficiently cleave pre-tRNA containing an Rp-thio-substitution at the scissile bond. These data are entirely consistent with the hypothesis that RNase P in plant chloroplasts is not a ribozyme, but a conventional protein enzyme.
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PMID:Spinach chloroplast RNase P: a putative protein enzyme. 864 12

We have studied variants of Escherichia coli RNase P RNA with base exchanges in the joining regions flanking helix P18, which form part of the ribozyme core structure. Mutant RNase P RNAs were analyzed for: (1) specific tRNA binding by gel retardation; (2) catalytic performance in single turnover reactions; (3) structural perturbations utilizing Pb2+ -induced hydrolysis; and (4) in vivo function by complementation analysis in E. coli RNase P mutant strains. Our in vitro experiments revealed that the base moieties of nucleotides (nt) 303 and 331 to 333 neither significantly contribute to tRNA binding or structural stabilization of RNase P RNA nor to active site chemistry. Single base exchanges at nt 300, 301, and 330 reduced tRNA binding, while having little effect on the catalytic rate, which demonstrates that these nucleotides are involved in forming the high affinity (pre-)tRNA binding site. In contrast, point mutations at the strictly conserved positions nt 328, 329, 334 and 335 reduced tRNA binding affinity as well as the catalytic rate, suggesting that these mutations additionally disrupted important interactions in the catalytic center. Probing by Pb2+ revealed that particularly the mutations that affected catalytic function most strongly perturbed a more extended region (nt 248 to 335) known to be involved in tRNA binding. Under high salt conditions (> or = 0.8 M NH4+), catalytic defects of the mutant RNase P RNAs were much less pronounced, suggesting that structural perturbations leading to increased electrostatic repulsion between phosphate groups were the main cause for observed functional defects. Only mutant C334 retained a largely increased pre-steady-state K(m(pss)) under high salt conditions. We conclude that the base at position 334 is directly involved in a contact crucial to pre-tRNA binding. A complementation analysis demonstrated the important role in vivo of the joining regions flanking helix P18. None of the bases could be mutated without affecting bacterial viability.
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PMID:Mutational analysis of the joining regions flanking helix P18 in E. coli RNase P RNA. 867 78

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.
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PMID:Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm. 875 10

Ribonuclease P (RNase P) is a ribonucleoprotein responsible for the endonucleolytic cleavage of the 5'-termini of tRNAs. Ribonuclease MRP (RNase MRP) is a ribonucleoprotein that has the ability to cleave both mitochondrial RNA primers presumed to be involved in mitochondrial DNA replication and rRNA precursors for the production of mature rRNAs. Several lines of evidence suggest that these two ribonucleoproteins are related to each other, both functionally and evolutionarily. Both of these enzymes have activity in the nucleus and mitochondria. Each cleave their RNA substrates in a divalent cation dependent manner to generate 5'-phosphate and 3'-OH termini. In addition, the RNA subunits of both complexes can be folded into a similar secondary structure. Each can be immunoprecipitated from mammalian cells with Th antibodies. In yeast, both have been found to share at least one common protein. This review will discuss some of the recent advances in our understanding of the structure, function and evolutionary relationship of these two enzymes in the yeast, Saccharomyces cerevisiae.
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PMID:The yeast, Saccharomyces cerevisiae, RNase P/MRP ribonucleoprotein endoribonuclease family. 890 93

Ribonuclease P (RNase P) is an essential enzyme whose action produces the mature 5' termini of all cellular and organellar transfer RNA molecules. In bacteria, the catalytic subunit of RNase P is an RNA molecule which by itself can bind substrate pre-tRNA, select and hydrolyze the correct phosphodiester bond, and release product tRNA. The simple requirements of the reaction-a monovalent cation such as K+ or NH4+ and the divalent cation Mg2+ (or Mn2+)-have prompted proposals that all aspects of phosphodiester bond hydrolysis might be accomplished by one or more divalent metal cations coordinated to the enzyme or substrate. To precisely localize the ligands of catalytically-involved Mg2+, we assayed cleavage by Escherichia coli RNase P RNA of pre-tRNA in which specific pro-Rp phosphate oxygens were replaced with sulfur. RNase P cleavage was targeted to that bond, at or nearest to the normal cleavage site, at which Mg2+ or Mn2+ could be coordinated. Single-turnover kinetics demonstrated that the apparent rate constant for the hydrolysis event was determined quantitatively by the affinity of the divalent cation (Mg2+ or Mn2+) for the atom (O or S) at the pro-Rp position of the scissile phosphodiester bond. We propose a model for pre-tRNA cleavage in which an essential Mg2+ ion is coordinated directly to the pro-Rp phosphate oxygen and indirectly to two other ligands near the scissile bond: the upstream ribose 2'-hydroxyl and the downstream purine N7. This catalytic Mg2+ ion most likely positions and deprotonates a water molecule for in-line nucleophilic attack on the scissile bond phosphorus.
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PMID:Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond. 905 47

The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. A self-cleaving RNase P RNA-substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2'-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2'-hydroxyl that, when substituted with 7-deazaadenine or 2'-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate-phosphate in the tertiary structure of the ribozyme-substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2'-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.
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PMID:Identification by modification-interference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P. 970 Dec 85

The bacterial RNase P ribozyme is a site-specific endonuclease that catalyzes the removal of pre-tRNA leader sequences to form the 5' end of mature tRNA. While several specific interactions between enzyme and substrate that direct this process have been determined, nucleotides on the ribozyme that interact directly with functional groups at the cleavage site are not well-defined. To identify individual nucleotides in the ribozyme that are in close proximity to the pre-tRNA cleavage site, we introduced the short-range photoaffinity cross-linking reagent 6-thioguanosine (s6G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence [tRNA(G-1)] and examined cross-linking in representatives of the two structural classes of bacterial RNase P RNA (from Escherichia coli and Bacillus subtilis). These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s6G upstream of the cleavage (-1) site is cleaved accurately. Interestingly, s6G at position +1 of tRNA cross-links with high efficiency to homologous positions in J5/15 in both E. coli and B. subtilis RNase P RNAs, while s6G at position -1 of tRNA(G-1) cross-links to homologous nucleotides in J18/2. Both cross-links are detected over a range of ribozyme and substrate concentrations, and importantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cleave the covalently attached substrate. These data indicate that the conserved guanosine at the 5' end of tRNA is adjacent to A248 (E. coli) of J5/15, while the base upstream of the substrate phosphate is adjacent to G332 (E. coli) of J18/2 and, along with available biochemical data, suggest that these nucleotides play a direct role in binding the substrate at the cleavage site.
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PMID:Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking. 986 Aug 78

Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3'-OH and 5'-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3'-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3'- S -phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3'- S -phosphorothiolate-modified ptRNA carrying a 7 nt 5'-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5'-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn(2+)or Cd(2+). To suppress aberrant cleavage, we also constructed a 3'- S -phosphorothiolate-modified ptRNA with a 1 nt 5'-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3'- S -phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.
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PMID:Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage. 1063 23

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.
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PMID:Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme. 1078 42

The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P.
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PMID:Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism. 1078 45


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