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)

Mobile group II introns encode multidomain proteins with maturase activity involved in splicing and reverse transcriptase (RT) and (often) endonuclease activities involved in intron mobility. These activities are present in a ribonucleoprotein complex that contains the excised intron RNA and the intron-encoded protein. Here, we report biochemical studies of the protein encoded by the group IIA1 intron in the cob gene of fission yeast Schizosaccharomyces pombe mitochondria (cobI1). RNP particle fractions from the wild-type fission yeast strain with cobI1 in its mtDNA have RT activity even without adding an exogenous primer. Characterization of the cDNA products of such reactions showed a strong preference for excised intron RNA as template. Two main regions for initiation of cDNA synthesis were mapped within the intron, one near the DIVa putative high-affinity binding site for the intron-encoded protein and the other near domain VI. Adding exogenous primers complementary to cob exon 2 sequences near the intron/exon boundary stimulated RT activity but mainly for pre-mRNA rather than mRNA templates. Further in vitro experiments demonstrated that cobI1 RNA in RNP particle fractions can reverse splice into double-stranded DNA substrates containing the intron homing site. Target DNA primed reverse transcription was not detected unless a DNA target was used that was already nicked in the antisense strand of exon 2. This study shows that S.pombe cobI1 encodes RNP particles that have most of the biochemical activities needed for it to be a retroelement. Interestingly, it appears to lack an endonuclease activity, suggesting that the active homing exhibited by this intron in crosses may differ somewhat from that of the better-characterized introns.
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PMID:Reverse transcriptase and reverse splicing activities encoded by the mobile group II intron cobI1 of fission yeast mitochondrial DNA. 1275 69

In bacteria, the tRNA-processing endonuclease ribonuclease P is composed of a large ( approximately 400 nucleotide) catalytic RNA and a smaller ( approximately 100 amino acid) protein subunit that is essential for substrate recognition. Current biochemical and biophysical investigations are providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate specificity and the role of essential metal ions in catalysis. Together with recent high-resolution structures of portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and protein are coordinated in this ribonucleoprotein enzyme.
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PMID:Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P. 1283 83

Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA. This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence. Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells. Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells. These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy. This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy.
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PMID:Engineering of RNase P ribozyme for gene-targeting applications. 1295 77

Group II intron-encoded proteins promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein complex. The Lactococcus lactis L1.LtrB intron encodes a maturase, LtrA, with reverse transcriptase homology and specific binding affinity for two domains of the intron RNA. The catalytically active ribonucleoprotein (RNP) has splicing, endonuclease, and reverse transcriptase activity, enabling efficient insertion of the intron sequence by a retro-homing mechanism. To determine the composition and assembly mechanism of the RNP complex, purified LtrA protein was analyzed for its ability to recognize a series of intron-derived RNAs. Equilibrium dissociation measurements show that LtrA recognizes two intronic domains, DI and DIV. However, distinct electrostatic requirements for binding imply different modes of molecular recognition in each case. Stoichiometric binding experiments show that the functional RNP complex consists of a dimeric protein species bound to a single intron RNA. Significant differences between the measured equilibrium dissociation constants and kinetically derived values suggest that conformational changes accompany assembly of the intron-maturase complex, and results of limited proteolysis and fluorescence spectroscopy experiments suggest that significant RNA-dependent structural changes within the maturase occur upon association with the intron. These results support a mutually induced fit model in which RNA-dependent conformational changes within LtrA enable stable association of the protein dimer with two independent intron domains to form a functional RNP.
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PMID:Assembly of an active group II intron-maturase complex by protein dimerization. 1515 82

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 A from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.
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PMID:Evolution from DNA to RNA recognition by the bI3 LAGLIDADG maturase. 1611 39

Non-long terminal repeat (LTR) retrotransposons are major components of the higher eukaryotic genome. Most of them have two open reading frames (ORFs): ORF2 encodes mainly the endonuclease and reverse transcriptase domains, but the functional features of ORF1 remain largely unknown. We used telomere-specific non-LTR retrotransposon SART1 in Bombyx mori and clarified essential roles of the ORF1 protein (ORF1p) in ribonucleoprotein (RNP) formation by novel approaches: in vitro reconstitution and in vivo/in vitro retrotransposition assays using the baculovirus expression system. Detailed mutation analyses showed that each of the three CCHC motifs at the ORF1 C terminus are essential for SART1 retrotransposition and are involved in packaging the SART1 mRNA specifically into RNP. We also demonstrated that amino acid residues 555 to 567 and 285 to 567 in the SART1 ORF1p are crucial for the ORF1p-ORF1p and ORF1p-ORF2p interactions, respectively. The loss of these domains abolishes protein-protein interaction, leading to SART1 retrotransposition deficiency. These data suggest that systematic formation of RNP composed of ORF1p, ORF2p, and mRNA is mainly mediated by ORF1p domains and is a common, essential step for many non-LTR retrotransposons encoding the two ORFs.
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PMID:Essential domains for ribonucleoprotein complex formation required for retrotransposition of telomere-specific non-long terminal repeat retrotransposon SART1. 1678

MicroRNAs (miRNAs) are a group of recently discovered small RNAs produced by the cell using a unique process, involving RNA polymerase II, Microprocessor protein complex, and the RNAase III/Dicer endonuclease complex, and subsequently sequestered in an miRNA ribonucleoprotein complex. The biological functions of miRNAs depend on their ability to silence gene expression, primarily via degradation of the target mRNA and/or translational suppression, mediated by the RNA-induced silencing complex (RISC). First discovered in Caenorhabditis elegans (lin-4), miRNAs have now been identified in a wide array of organisms, including plants, zebrafish, Drosophila, and mammals. The expression of miRNAs in multicellular organisms exhibits spatiotemporal, and tissue- and cell-specificity, suggesting their involvement in tissue morphogenesis and cell differentiation. More than 200 miRNAs have been identified or predicted in mammalian cells. Recent studies have demonstrated the importance of miRNAs in embryonic stem cell differentiation, limb development, adipogenesis, myogenesis, angiogenesis and hematopoiesis, neurogenesis, and epithelial morphogenesis. Overexpression (gain-of-function) and inactivation (loss-of-function) are currently the primary approaches to studying miRNA functions. Another family of small RNAs related to miRNAs is the small interfering RNAs (siRNAs), generated by Dicer from long double-stranded RNAs (dsRNAs), and produced from an induced transgene, a viral intruder, or a rogue genetic element. siRNAs silence genes via either mRNA degradation, using the RISC, or DNA methylation. siRNAs are actively being applied in basic, functional genetic studies, particularly in the generation of gene knockdown animals, as well as in gene knockdown studies of cultured cells. These studies have provided invaluable information on the specific function(s) of individual genes. siRNA technology also presents exciting potential as a therapeutic approach in disease prevention and treatment, as suggested by a recent study targeting apolipoprotein B (ApoB) in primates. Further elucidation of how miRNAs and other small RNAs interact with known and yet-to-be identified gene regulatory pathways in the cell should provide us with a more in-depth understanding of the mechanisms regulating cellular function and differentiation, and facilitate the application of small RNA technology in disease control and treatment.
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PMID:MicroRNAs and cell differentiation in mammalian development. 1684 91

LINE-1 or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high-affinity RNA-binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition.
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PMID:The ORF1 protein encoded by LINE-1: structure and function during L1 retrotransposition. 1687 16

Ribonuclease P (RNase P) is a ubiquitous endonuclease that catalyses the maturation of the 5' end of transfer RNA (tRNA). Although it carries out a biochemically simple reaction, RNase P is a complex ribonucleoprotein particle composed of a single large RNA and at least one protein component. In bacteria and some archaea, the RNA component of RNase P can catalyse tRNA maturation in vitro in the absence of proteins. The discovery of the catalytic activity of the bacterial RNase P RNA triggered numerous mechanistic and biochemical studies of the reactions catalysed by the RNA alone and by the holoenzyme and, in recent years, structures of individual components of the RNase P holoenzyme have been determined. The goal of the present review is to summarize what is known about the bacterial RNase P, and to bring together the recent structural results with extensive earlier biochemical and phylogenetic findings.
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PMID:Bacterial RNase P: a new view of an ancient enzyme. 2128 79

Ribonuclease P (RNase P) is the ribonucleoprotein endonuclease that processes the 5' ends of precursor tRNAs. Bacterial and eukaryal RNase P RNAs had the same primordial ancestor; however, they were molded differently by evolution. RNase P RNAs of eukaryotes, in contrast to bacterial RNAs, are not catalytically active in vitro without proteins. By comparing the bacterial and eukaryal RNAs, we can begin to understand the transitions made between the RNA and protein-dominated worlds. We report, based on crosslinking studies, that eukaryal RNAs, although catalytically inactive alone, fold into functional forms and specifically bind tRNA even in the absence of proteins. Based on the crosslinking results and crystal structures of bacterial RNAs, we develop a tertiary structure model of the eukaryal RNase P RNA. The eukaryal RNA contains a core structure similar to the bacterial RNA but lacks specific features that in bacterial RNAs contribute to catalysis and global stability of tertiary structure.
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PMID:Structure and function of eukaryotic Ribonuclease P RNA. 1708 93


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