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)

The tRNATyr precursor molecule, synthesized from phi 80 psu3+ DNA (containing a single tRNA gene) by DNA-dependent RNA polymerase and q factor, was about 205 nucleotides long. The main product of its digestion with a ribonuclease tii preparation from Escherichia coli showed the same electrophoretic mobility as tRNAtyr precursor isolated in vivo and was found to be identical to it when analysed using fingerprint techniques. This intermediate precursor synthesized in vitro was converted further by processing with ribonuclease P into an RNA identical size to mature tRNATyr. It was concluded that the initiation of transcription of the tRNATyr gene in vitro occurs at the same site as that of transcription in vivo and a termination occurs at about 80 nucleotides beyond the CCA end of tRNATyr.
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PMID:Processing by ribonuclease II of the tRNATyr precursor of Escherichia coli synthesized in vitro. 32 7

Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 1000000 x g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3'-terminal ends of monomeric tRNA precursors. Some properties of these two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease P, a well-characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.
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PMID:Specific ribonucleases involved in processing of tRNA precursors of Escherichia coli. Partial purification and some properties. 35 May 82

We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.
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PMID:In vitro synthesis of transfer RNA. I. Purification of required components. 109 89

We have shown that the synthesis of active su+III tRNATyr from a phi80psu+III DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-dependent RNA polymerase, catalyzes the formation of a large molecular weight transcript, initiating synthesis at a specific site 41 nucleotides proximal to the 5' end of the su+III tRNATyr structural gene and continuing at least 100 nucleotides beyond the 3' terminus of the su+III tRNATyr sequence. The second required component, designated Fraction V, allows purified DNA-DEPENDENT RNA polymerase to function in tRNA synthesis. We have shown that this fraction contains an endonuclease that together with DNA-dependent RNA polymerase is responsible for the synthesis of su+III tRNATyr "precursor". Thus, su+III tRNATyr precursor is not itself the primary transcription product of the su+III tRNATyr gene, but rather, it arises as a result of post-transcriptional cleavage of a much larger transcript by the action of the nuclease present in Fraction V. The third enzymatic activity required for synthesis of active su+III tRNATyr is a ribonuclease (RNase P III) that specifically catalyzes the removal of the 3' extra nucleotides from the su+III tRNATyr precursor. The fourth activity required for synthesis of tRNA is a previously identified endonuclease, RNase P, that specifically catalyzes the removal of the 5' extra nucleotides from tRNA precursors. The properties of RNase P purified according to the procedure developed in this laboratory have been compared with those of the enzyme purified from ribosomes according to the procedure described by Robertson et al. (Robertson, H.D., Altman, S., and Smith, F.D. (1972) J.Biol. Chem. 247, 5243-5251.).
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PMID:In vitro synthesis of transfer RNA. II. Identification of required enzymatic activities. 109 90

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function. 752 70

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.
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PMID:Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease. 761 61

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.
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PMID:Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA. 800 21

The ribozyme ribonuclease (RNase) P cleaves precursor transcripts to produce the mature 5'-end of tRNAs. This hydrolysis reaction has a divalent cation requirement that is primarily catalytic, rather than structural; RNase P can be considered a metalloenzyme. Kinetic analysis shows that the RNase P catalytic mechanism has a cooperative dependence upon Mg2+ concentration. At least three Mg2+ ions are required for optimal activity, suggesting a multiple metal ion mechanism. The 2'-OH at the site of substrate cleavage may act as a ligand for a catalytically important Mg2+: deoxyribose substitution reduces the apparent number of Mg2+ bound from three to two and increases the apparent dissociation constant for Mg2+ from the micromolar to the millimolar range. In addition to these cation effects, the deoxyribose substitution reduces the rate of catalysis by 3400-fold; substitution with 2'-O-methyl at the cleavage site reduces the catalytic rate 10(6)-fold. If we presume no significant conformational effects of the substitutions, these results suggest that the 2'-OH serves as hydrogen-bond donor. The kinetic analysis of the catalytic mechanism is based upon the characterization of the pH dependence of the reaction. There is a hyperbolic (saturable) dependence on hydroxide concentration, with the half-maximal rate achieved at pH 8.0-8.5. The rate of the cleavage step is about 200 min-1 at pH 8.0, which is 500-fold faster than the steady-state parameter kcat.
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PMID:Multiple magnesium ions in the ribonuclease P reaction mechanism. 849 32

Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole-cell extract as the starting material. A 4400-fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA-affinity column chromatography. This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety. These seven polypeptides consistently copurified with the RNase P activity through two ion-exchange chromatography columns and in a glycerol gradient. As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity. The 3'-end-labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5' end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P. The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8-bp acceptor stem. This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position -1) of tRNAHis in A. nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts.
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PMID:Purification and characterization of mitochondrial ribonuclease P from Aspergillus nidulans. 863 44

Several RNA molecules that copurified with Aspergillus nidulans mitochondrial ribonuclease (RNase) P were identified [Lee, Y C., Lee, B. J., Hwang, D. S. & Kang, H. S. (1996) Eur J. Biochem. 235, 289-296], and their partial sequences were determined. Using an oligonucleotide probe, we cloned and mapped the gene encoding this putative RNA component of RNase P (RNase P-RNA), situated between URFA3 (unidentified reading frame A3) and cobA (apocytochrome b) genes in the mitochondrial genome of A. nidulans. The gene is extremely (A+T)-rich and contains two regions of sequence similarity conserved among the known mitochondrial RNase P-RNAs and the eubacterial RNase P-RNAs. The determination of 5' and 3' termini by primer extension and sequencing indicated that the length of the RNA transcript is 232 nucleotides. Northern-blot analysis revealed that its only subcellular location was the mitochondria. Two RNase P-RNA fragments of 110 nucleotides and 80 nucleotides, each containing one of the two conserved regions, could be recovered from the nuclease-treated enzyme without significant loss of activity. The sizes of these fragments appeared to be the minimum lengths required for the vitro activity of the enzyme.
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PMID:The RNA component of mitochondrial ribonuclease P from Aspergillus nidulans. 863 45


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