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)

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.
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PMID:Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. 880 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

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

RNA editing in trypanosome mitochondria entails the posttranscriptional internal addition and occasional deletion of uridines from precursor mRNAs. Ample evidence exists to show that the information specifying the site and number of uridines added or deleted comes from small, mitochondrially encoded guide RNAs (gRNAs). More recent work indicates that the process involves an enzymatic cascade, initiating with an endonucleolytic cleavage of the pre-mRNA at an editing site. The cleaved editing site can undergo uridine (U) addition to or deletion from the 3' end of the 5' fragment via a mitochondrial terminal uridylyl transferase (TUTase) or terminal uridylyl exonuclease, respectively. Mitochondrial RNA ligase subsequently rejoins the mRNA. Activities to carry out these processes have been found in trypanosome mitochondria, including an editing-site-specific endonuclease activity which cleaves preedited but not edited mRNAs. We have found that this enzymatic activity cosediments with the same 19S ribonucleoprotein particle previously shown to contain TUTase, RNA ligase, and gRNAs and remains stable after salt treatment. Depletion of endogenous cytochrome b gRNAs by the addition of complementary oligonucleotides in vitro completely inhibits editing-site cleavage of synthetic preedited cytochrome b mRNA. The addition of synthetic cognate gRNA for cytochrome b but not unrelated small RNA restores editing-site cleavage. These studies show that in addition to specifying the site and number of uridines added or deleted, gRNAs provide the necessary information for cleavage by the editing-site-specific endonuclease.
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PMID:Guide RNA requirement for editing-site-specific endonucleolytic cleavage of preedited mRNA by mitochondrial ribonucleoprotein particles in Trypanosoma brucei. 927 15

Group II introns use intron-encoded reverse transcriptase, maturase and DNA endonuclease activities for site-specific insertion into DNA. Remarkably, the endonucleases are ribonucleoprotein complexes in which the excised intron RNA cleaves the sense strand of the recipient DNA by reverse splicing, while the intron-encoded protein cleaves the antisense strand. Here, studies with the yeast group II intron aI2 indicate that both the RNA and protein components of the endonuclease contribute to recognition of an approximately 30 bp DNA target site. Our results lead to a model in which the protein component first recognizes specific nucleotides in the most distal 5' exon region of the DNA target site (E2-21 to -11). Binding of the protein then leads to DNA unwinding, enabling the intron RNA to base pair to a 13 nucleotide DNA sequence (E2-12 to E3+1) for reverse splicing. Antisense-strand cleavage requires additional interactions of the protein with the 3' exon DNA (E3+1 to +10). Our results show how enzymes can use RNA and protein subunits cooperatively to recognize specific sequences in double-stranded DNA.
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PMID:Group II intron endonucleases use both RNA and protein subunits for recognition of specific sequences in double-stranded DNA. 936 97

Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.
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PMID:Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis. 966 81

The retrohoming of the yeast mtDNA intron aI1 occurs by a target DNA-primed reverse transcription (TPRT) mechanism in which the intron RNA reverse splices directly into the recipient DNA and is then copied by the intron-encoded reverse transcriptase. Here, we carried out biochemical characterization of the intron-encoded reverse transcriptase and site-specific DNA endonuclease activities required for this process. We show that the aI1 reverse transcriptase has high TPRT activity in the presence of appropriate DNA target sites, but differs from the closely related reverse transcriptase encoded by the yeast aI2 intron in being unable to use artificial substrates efficiently. Characterization of TPRT products shows that the fully reverse spliced intron RNA is an efficient template for cDNA synthesis, while reverse transcription of partially reverse spliced intron RNA is impeded by the branch point. Novel features of the aI1 reaction include a prominent open-circular product in which cDNAs are incorporated at a nick at the antisense-strand cleavage site. The aI1 endonuclease activity, which catalyzes the DNA cleavage and reverse splicing reactions, is associated with ribonucleoprotein particles containing the intron-encoded protein and the excised intron RNA. As shown for the aI2 endonuclease, both the RNA and protein components are used for DNA target site recognition, but the aI1 protein has less stringent nucleotide sequence requirements for the reverse splicing reaction. Finally, perhaps reflecting this relaxed target specificity, in vitro experiments show that aI1 can reverse splice directly into ectopic mtDNA transposition sites, consistent with the previously suggested possibility that this mechanism is used for ectopic transposition of group II introns in vivo.
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PMID:Group II intron mobility in yeast mitochondria: target DNA-primed reverse transcription activity of aI1 and reverse splicing into DNA transposition sites in vitro. 973 19

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.
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PMID:RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. 975 84

Ribonuclease P is the endonuclease required for generating the mature tRNA 5'-end. The ribonucleoprotein character of this enzyme has now been proven in most organisms and organelles. Exceptions, however, are still the chloroplasts, plant nuclei and animal mitochondria where no associated RNAs have been detected to date. In contrast to the known RNA subunits, which are fairly well-conserved in size and structure among diverse phylogenetic groups, the protein contribution to the holoenzyme is highly variable in size and number of the individual components. The structure of the bacterial protein component has recently been solved. In contrast, the spatial arrangement of the multiple subunits in eukaryotic enzymes is still enigmatic. Substrate requirements of the enzymes or their catalytic RNA subunits are equally diverse, ranging from simple single domain mimics to an almost intact three-dimensional structure of the pre-tRNA substrate. As an example for an intermediate in the enzyme evolution, ribonuclease P from the Cyanophora paradoxa cyanelle will be discussed in more detail. This enzyme is unique, as it combines cyanobacterial and eukaryotic features in its function, subunit composition and holoenzyme topology.
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PMID:Ribonuclease P: the diversity of a ubiquitous RNA processing enzyme. 1037 Oct 40

Self-splicing group II introns may be the evolutionary progenitors of eukaryotic spliceosomal introns, but the route by which they invade new chromosomal sites is unknown. To address the mechanism by which group II introns are disseminated, we have studied the bacterial L1.LtrB intron from Lactococcus lactis. The protein product of this intron, LtrA, possesses maturase, reverse transcriptase and endonuclease enzymatic activities. Together with the intron, LtrA forms a ribonucleoprotein (RNP) complex which mediates a process known as retrohoming. In retrohoming, the intron reverse splices into a cognate intronless DNA site. Integration of a DNA copy of the intron is recombinase independent but requires all three activities of LtrA. Here we report the first experimental demonstration of a group II intron invading ectopic chromosomal sites, which occurs by a distinct retrotransposition mechanism. This retrotransposition process is endonuclease-independent and recombinase-dependent, and is likely to involve reverse splicing of the intron RNA into cellular RNA targets. These retrotranspositions suggest a mechanism by which splicesomal introns may have become widely dispersed.
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PMID:Retrotransposition of a bacterial group II intron. 1080 Nov 7


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