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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The leuX gene of Escherichia coli codes for a suppressor tRNA and forms a single gene operon containing its own promoter and Q-independent terminator. An analysis of the in vitro processing of leuX precursor revealed that the processing of the 5' end took place in a single-step reaction catalysed by RNase P while the 3' processing involved two successive reactions. The endonucleolytic cleavage activity of the 3' precursor sequence was found to copurify with RNase P. Heat inactivation of thermosensitive RNase P from two independent E. coli mutants abolished the cleavage activity of both the 5' and 3' ends. These results altogether suggest that RNase P carries the activity of 3' end cleavage as well as that of 5' processing. In the presence of Mg2+ alone, the leuX precursor was found to be self-cleaved at a site approximately 13 nt inside from the 5' end of mature tRNA. The self-cleaved precursor tRNA was no longer processed by the 3' endonuclease, suggesting that the 3' endonuclease recognizes a specific conformation of the precursor tRNA for action.
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PMID:A novel function of RNase P from Escherichia coli: processing of a suppressor tRNA precursor. 306 98

An endonuclease activity was isolated from 100,000 g supernatant fraction of Escherichia coli using in vitro primary transcripts of T4 tRNA gene cluster as assay substrates. The endonuclease cleaves the polycistronic RNA precursors into fragments containing monomeric and dimeric stable RNA sequences. The result strongly suggest that this enzyme participates in the early steps of T4 tRNA maturation pathway preceding the action of RNase P.
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PMID:An Escherichia coli endonuclease responsible for primary cleavage of in vitro transcripts of bacteriophage T4 tRNA gene cluster. 625 21

The seven tRNA genes clustered in the supB-E region of the Escherichia coli chromosome were transcribed in vitro with purified RNA polymerase, using a restriction fragment from lambda psu degrees 2, a transducing phage carrying the chromosome region, as template. A single major transcript was synthesized, which was about 770 nucleotides long and contained all seven tRNA sequences. The terminal sequences of the transcript were determined and mapped on the DNA sequence of the supB-E region previously determined. The transcription start site is seven base pairs downstream from the Pribnow box sequence, as expected from the DNA sequence analysis and consistent with the findings on the trimeric tRNA precursor (pppG--tRNAMETM-tRNALeu-tRNAGln1) which was detected in an RNase P mutant and shown to be coded for by the supB-E region. Cleavage of the restriction fragment at the -35 region with another restriction endonuclease abolished the template activity of the fragment. Transcription of the supB-E tRNA operon was relatively unaffected by the presence of rho factor. Transcription termination occurs within a region of three bases between positions 770 and 772 from the transcription start site. Immediately upstream from the termination sites, there is a region of 26 nucleotides that could form a stem structure, thereby consistent with the general feature of rho-independent termination sites.
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PMID:In vitro transcription of the supB-E tRNA operon of Escherichia coli. Characterization of transcription products. 628 82

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.
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PMID:Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA. 752 Dec 97

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3'-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5'- and 3'-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5'-end precedes that at the 3'-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5'-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.
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PMID:Human mitochondrial tRNA processing. 775 47

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

Ribonuclease P (RNaseP) generates the mature 5' end of tRNAs by removing 5'leader sequences from pre-tRNAs. In vitro, the RNA subunit is sufficient to catalyze this reaction and is therefore a ribozyme. The kinetic analysis of RNase P-mediated catalysis is complicated because product release is normally rate-limiting. Furthermore, the intermolecular nature of the cleavage reaction precludes many applications of in vitro selection schemes to the analysis of RNaseP. To examine and manipulate the RNase P function more effectively, we designed a pair of ribozymes in which the RNase P RNA is covalently linked to a pre-tRNA substrate. To facilitate intramolecular cleavage, pre-tRNA molecules were tethered to circulatory permuted RNaseP RNA molecules at nucleotides implicated in substrate binding. These "active-site-tethered" pre-tRNA-RNaseP RNA conjugates undergo accurate and efficient self-cleavage in vitro, with first-order reaction rates equivalent to the rate of the chemical step of the native RNase P reaction. Unlike most ribozymes, RNase P recognizes its substrate through tertiary RNA-RNA interactions, rather than through extensive Watson-Crick base-pairing. However, the development of the active-site-tethered conjugates has led us to create a sequence-specific endonuclease, termed Endo.P. In the Endo.P configuration, the 3'half of the pre-tRNA acceptor stem binds exogenous RNA substrates via Watson-Crick base-pairing; the bound substrate is subsequently cleaved at the predicted site. The demonstration of sequence-specific cleavage by Endo.P expands the potential of RNase P and its derivatives as reagents in gene therapy.
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PMID:Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates. 807 82

RNase P, the enzyme response for 5'-end processing of tRNAs and 4.5S RNA, has been extensively characterized from E. coli. The RNA component of E. coli RNase P, without the protein, has the enzymatic activity and is the first true RNA enzyme to be characterized. RNase P and MRP are two distinct nuclear ribonucleoprotein (RNP) particles characterized in many eukaryotic cells including human, yeast and plant cells. There are many similarities between RNase P and MRP. These include: (1) sequence specific endonuclease activity; (2) homology at the primary and secondary structure levels; and (3) common proteins in both the RNPs. It is likely that RNase P and MRP originated from a common ancestor.
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PMID:Structural and functional similarities between MRP and RNase P. 890 92

Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5' endonuclease, a 3' endonuclease, and a tRNA:CCA nucleotidyltransferase. The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors. Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA.protein complex as in Bacteria, Archaea, and Eukarya. The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein. Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg2+ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain.
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PMID:Structure, mechanism and evolution of chloroplast transfer RNA processing systems. 890 2

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.
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PMID:Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking. 917 92


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