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)

Argonaute proteins participate in conferring all known functions of RNA-mediated gene silencing phenomena. However, prior to structural investigations of this evolutionarily conserved family of proteins, there was little information concerning their mechanisms of action. Here, we describe our crystallographic analysis of the PIWI domain of an archaeal Argonaute homolog, AfPiwi. Our structural analysis revealed that the Argonaute PIWI fold incorporates both an RNase-H-like catalytic domain and an anchor site for the obligatory 5' phosphate of the RNA guide strand. RNA-AfPiwi binding assays combined with crystallographic studies demonstrated that AfPiwi interacts with RNA via a conserved region centered on the carboxyl terminus of the protein, utilizing a novel metal-binding site. A model of the PIWI domain of Argonaute in complex with a small interfering RNA (siRNA)-like duplex is consistent with much of the existing biochemical and genetic data, explaining the specificity of the RNA-directed RNA endonuclease reaction and the importance of the 5' region of microRNAs (miRNAs) (the "seed") to nucleate target RNA recognition and provide high-affinity guide-target interactions.
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PMID:Molecular mechanism of target RNA transcript recognition by Argonaute-guide complexes. 1738 Dec 79

Type II restriction-modification (R-M) systems are composed of linked restriction endonuclease and modification methyltransferase genes and serve as barriers to horizontal gene transfer even though they are mobile in themselves. Their products kill host bacterial cells that have lost the R-M genes, a process that helps to maintain the frequency of the R-M systems in the viable cell population. Their establishment and maintenance in a bacterial host are expected to involve fine regulation of their gene expression. In the present study, we analyzed transcription of the modification gene and its regulation within the EcoRI R-M system. Northern blotting revealed that the downstream ecoRIM gene is transcribed as a monocistronic mRNA and as part of a larger bicistronic mRNA together with the upstream ecoRIR gene. Primer extension, RNase protection, and mutational analysis using lacZ gene fusions identified two overlapping promoters for ecoRIM gene transcription within the ecoRIR gene. Further mutational analysis revealed that two upstream AT-rich elements within the ecoRIR gene, "AATAAA" and "ATTATAAATATA," function as negative regulators of these promoters. Simultaneous substitution of these two elements resulted in a four-fold increase in beta-galactosidase activity and a five-fold increase in transcript levels as measured by RNase protection assay. RNA measurements of the ecoRIM transcript suggested that these elements decreased ecoRIM expression by interfering with transcription initiation of the ecoRIM promoters. Possible roles for these ecoRIM promoters and their negative regulators in the EcoRI R-M system are discussed.
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PMID:Regulation of the EcoRI restriction-modification system: Identification of ecoRIM gene promoters and their upstream negative regulators in the ecoRIR gene. 1761 69

We have found novel functions of scaffold attachment factor-B1 (SAFB) during apoptosis. The experiments showed that SAFB moved into the nucleolus 15 min after the induction of apoptosis and before the release of cytochrome c into the cytoplasm. Two hours later SAFB formed a peri-nucleolar ring-like structure and this occurred after cytochrome c release and before PARP cleavage. Digestion with RNase suggested that the peri-nucleolar ring structure was dependent on RNA integrity and a RNA moiety formed part of this structure. Studies using SAFB deletion mutants showed that the formation of the peri-nucleolar structure was not mediated by the DNA binding (SAP) or the RNA binding (RRM) domain of SAFB but was instead dependent on the S/K and R/E coiled-coil regions: a result suggesting that the structure is formed via protein interactions. In addition, SAFB cleavage was shown to be mediated by caspase-3 and occurred after the formation of the peri-nucleolar ring and after cleavage of PARP (characteristic of proteins having a direct role in apoptosis). A determinant for this cleavage is located in the DNA binding domain and we hypothesize that SAFB may direct the reorganization and segregation of nuclear RNA and DNA prior to endonuclease-mediated DNA cleavage.
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PMID:SAFB re-distribution marks steps of the apoptotic process. 1764 27

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.
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PMID:Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture. 1788 80

4'-thio DNA consisting of 2'-deoxy-4'-thionucleosides exhibits resistance to both endonuclease and 3'-exonuclease cleavages. Interestingly, we found that 4'-thioDNA duplex behaved like RNA molecules in hybridization properties and structural aspects. Here, we have determined the structure of 4'-thioDNA duplex in solution by NMR. Most residues take on C3'-endosugar puckering, which is characteristic to A-form. The major groove of 4'-thioDNA duplex is narrow and deep, while the minor groove is wide and shallow. These features are also characteristic to A-form. Thus, although DNA duplex usually takes on B-form in solution, 4'-thioDNA takes on A-form, like RNA molecules. A-form-like groove features of 4'-thioDNA can account for the fact that 4'-thioDNA duplex interacts with an RNA major groove binder, but not with DNA groove binder. Interaction of 4'-thioDNA with RNase V1 and resistance to endonuclease may also be accounted for in the same context.
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PMID:Structure of 4'-thioDNA which exhibits endonuclease resistance. 1802 26

The genomic region of Prevotella bryantii TC1-1 that conferred an increased nucleolytic activity on Escherichia coli was characterized. It contains two divergent transcriptional units separated by an AT-rich promoter region. One unit is comprised of three genes involved in nucleotide metabolism. nucA, the first gene of this unit, whose product belongs to exonuclease/endonuclease/phosphatase Pfam family, was thought to be required for the increased nucleolytic activity and various expression strategies were employed to confirm its role. The nucA expression was only successful in cell free system where DNase and RNase activity was observed. Two genes downstream of nucA code for a putative uracil DNA glycosylase and uridine kinase which could be involved in the removal of misincorporated uracil from DNA and its reuse. Given that apurinic/apyrimidinic nuclease activity is required after uracil removal from DNA, it was somewhat surprising to find out that nucA, whose product belongs to protein family consisting mostly of apurinic/apyrimidinic nucleases, has no apurinic/apyrimidinic activity.
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PMID:Expression of nuclease gene nucA, a member of an operon putatively involved in uracil removal from DNA and its subsequent reuse in Prevotella bryantii. 1842 83

The 140-nucleotide trp leader RNA, which is formed by transcription termination under conditions of high intracellular tryptophan, was used to study RNA turnover in Bacillus subtilis. We showed in vivo that the amount of endonuclease cleavage at approximately nucleotide 100 is decreased under conditions where RNase J1 concentration is reduced. In addition, under these conditions the level of 3'-terminal RNA fragments, which contain the strong transcription terminator structure, increases dramatically. These results implicated RNase J1 in the initiation of trp leader RNA decay as well as in the subsequent steps leading to complete turnover of the terminator fragment. To confirm a direct role for RNase J1, experiments were performed in vitro with various forms of trp leader RNA and 3'-terminal RNA fragments. Specific endonuclease cleavages, which were restricted to single-stranded regions not bound by protein, were observed. Degradation of the 3'-terminal fragment by the 5' to 3'-exonuclease activity of RNase J1 was also demonstrated, although the presence of strong secondary structure impeded RNase J1 processivity to some extent. These results are consistent with a model for mRNA decay in Bacillus subtilis whereby the downstream products of RNase J1 endonucleolytic cleavage become substrates for the 5' to 3'-exoribonuclease activity of the enzyme.
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PMID:Role of Bacillus subtilis RNase J1 endonuclease and 5'-exonuclease activities in trp leader RNA turnover. 1844 92

Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation.
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PMID:Hypernegative supercoiling inhibits growth by causing RNA degradation. 1879 Aug 62

Individual transfer ribonucleic acids (tRNAs) in a complex mixture can be identified by the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detection of their signature digestion products. Signature digestion products are endonuclease digestion products whose mass-to-charge value is unique thus corresponding to only a single tRNA. To improve the effectiveness of this approach, we have expanded the applicable endonucleases and examined the use of multiple endonucleases for tRNA identification. The purine specific endonucleases RNase T1 and RNase TA generate the largest number of predicted signature digestion products. Experimentally, MALDI-MS analysis of endonuclease digests from Escherichia coli and Bacillus subtilis finds that any two endonucleases used in combination increases tRNA identification by about 25% over the number identified with a single endonuclease. Using three endonucleases, RNase T1, RNase A, and RNase TA, further improves the number of tRNAs identified by 10-15% over those found with two endonucleases. Limitations in the MALDI-MS approach for complex mixtures were revealed in this study, suggesting that the direct MALDI-MS analysis of signature digestion products is more effective for organisms having 30 or less unique tRNAs.
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PMID:Multiple endonucleases improve MALDI-MS signature digestion product detection of bacterial transfer RNAs. 1910 81

Bacteriophage T4 is the archetype of virulent phage. It has evolved very efficient strategies to subvert host functions to its benefit and to impose the expression of its genome. T4 utilizes a combination of host and phage-encoded RNases and factors to degrade its mRNAs in a stage-dependent manner. The host endonuclease RNase E is used throughout the phage development. The sequence-specific, T4-encoded RegB endoribonuclease functions in association with the ribosomal protein S1 to functionally inactivate early transcripts and expedite their degradation. T4 polynucleotide kinase plays a role in this process. Later, the viral factor Dmd protects middle and late mRNAs from degradation by the host RNase LS. T4 codes for a set of eight tRNAs and two small, stable RNA of unknown function that may contribute to phage virulence. Their maturation is assured by host enzymes, but one phage factor, Cef, is required for the biogenesis of some of them. The tRNA gene cluster also codes for a homing DNA endonuclease, SegB, responsible for spreading the tRNA genes to other T4-related phage.
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PMID:RNA processing and decay in bacteriophage T4. 1921 70


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