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 RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from 5'-ends of pre-tRNAs. A photoaffinity cross-linking agent was coupled to the substrate phosphate on which RNase P acts and used to map nucleotides in the vicinity of the catalytic site of this ribozyme. Mature tRNA(Phe) containing a 5'-thiophosphate was synthesized by transcription in vitro using phage T7 RNA polymerase in the presence of guanosine 5'-phosphorothioate. The photoagent (azidophenacyl) was coupled uniquely to the 5'-thiophosphate of the tRNA, the site of action by RNase P. The photoagent-containing tRNA binds to RNase P RNA and is cross-linked by UV irradiation to it at high efficiency (10-30%). Cross-linked conjugates are enzymatically inactive, consistent with the occupancy of the active site of the RNase P RNA by the tRNA. Reversal of the cross-link by phenylmercuric acetate restores activity. The sites of cross-linking in RNase P RNA were determined by primer extension. In order to identify generalities and detect idiosyncrasies, analyses were carried out using RNase P RNAs from three phylogenetically diverse organisms: Bacillus subtilis, Chromatium vinosum and Escherichia coli. In the context of a phylogenetic structure model, two regions of cross-linking are observed in all three RNAs. Two of the RNAs cross-link to a lesser extent at a third structural region and one of the RNAs is cross-linked to a small extent to a fourth region. All the sites of cross-linking between the substrate phosphate in tRNA and the RNase P RNAs are in the conserved core of the structure model, consistent with the importance of the cross-linked residues to the action of this RNA enzyme.
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PMID:Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. 170 Nov 42

The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.
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PMID:Lead(II) cleavage analysis of RNase P RNA in vivo. 1604 96

With the exception of a few genes, most of the mitochondrial (mt) genome of Pneumocystis carinii has not previously been sequenced. Shotgun sequences generated as a result of the Pneumocystis Genome Project (PGP) were assembled with the gap4 assembly program into a 23-kb contig. Annotation of the mt genome identified 4 open reading frames and 20 tRNAs in addition to 17 other genes: ATP synthase, subunits 6, 8, and 9; cytochrome c oxidase, subunits 1, 2, and 3; NADH dehydrogenase, subunits 1, 2, 3, 4, 4L, 5, and 6; apocytochrome b; RNase P RNA gene; and the mitochondrial large and small ribosomal RNA subunits. A 24-bp unit that repeated from one to five times was identified interior to the ends of the mt genome. Migration of the genome on CHEF gels was consistent with that of linear DNA and digestion with BAL31 showed a concomitant reduction in size of the genome, a characteristic of linear DNA. Together with the identification of terminal repeats similar to those found in other linear fungal mt genomes and the inability to join the ends by PCR, these data provide strong evidence that the mt genome of P. carinii is linear.
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PMID:Sequence and structure of the linear mitochondrial genome of Pneumocystis carinii. 1992 Dec 62

The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed 'Lead-seq', provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5'-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.
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PMID:Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions. 3246 49