Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The small prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi 29 is essential for ATP-dependent packaging of viral DNA. The 174-, 124-, and 120-residue forms of pRNA produced in vitro using T7 RNA polymerase were equivalent in prohead binding and DNA packaging activity to pRNAs produced in phi 29-infected cells. pRNA binding to proheads, characterized by the use of Northern hybridization and filter binding assays, was specific, rapid, and irreversible in the presence of 10 mM Mg2+. Proheads produced in phage-infected cells carried 5.8 +/- 2.7 copies of pRNA, and proheads assembled in Escherichia coli in the absence of pRNA bound 6.0 +/- 3.5 copies of pRNA. Footprints of proheads on pRNA generated with the ribonucleases A, T1, and V1 showed that nucleotides 22-84, 5' to 3', were protected from ribonuclease attack. Enhanced cleavage at nucleotides 37-40 with ribonuclease V1 suggested a conformational change of pRNA upon prohead binding.
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PMID:Characterization of the prohead-pRNA interaction of bacteriophage phi 29. 810 96

The conformation of the last 201 nucleotides located at the 3'-end of brome mosaic virus (BMV) RNAs was investigated in solution using different chemical and enzymatic probes. Bases were probed with dimethylsulfate (which methylates N-1 positions of A, N-3 positions of C and N-7 positions of G), a carbodiimide (which modifies N-1 positions of G and N-3 positions of U) and diethylpyrocarbonate (which modifies N-7 positions of A). Ribonucleases T1, U2 and S1 were used to map unpaired nucleotides and ribonuclease V1 to monitor paired bases or stacked nucleotides. Cleavage or modification sites were detected by gel electrophoresis either indirectly by analyzing DNA sequence patterns generated by primer extension with reverse transcriptase of the modified RNAs or by direct identification within the statistical cleavage patterns of the RNA. On the basis of these biochemical results, an atomic model was built by computer modeling and its stereochemistry refined. The deduced secondary structure of the RNA confirms data previously proposed by others but contains additional base-pairs (A27-U32, A28-G31, G41-A134, G64-C68, U80-A99, G81-A98, G88-U91, G100-U126, U104-U125, G162-G166 and A172-A191), one new tertiary long-range interaction (U103-U164) and a small triple helical conformation with (G41-A134)-A18 and (C42-G133)-A17 interactions. The new secondary structure also indicates the existence of a second pseudoknot involving pairing between residues A181 to A184 and residues U197 to U194, outside the domain conferring tyrosylation ability to BMV RNA. The main outcome from the model stems from its intricate folding, which allows a new assignment for the domains mimicking the anticodon- and D-loop regions of tRNA. Interestingly, the stem and loop region found structurally to be analogous to the anticodon arm of tRNA(Tyr) does not contain the tyrosine anticodon involved in the aminoacylation process. The structural analogies with canonical tRNA(Tyr) illustrate the functional mimicry existing between the BMV RNA structure and canonical tRNA(Tyr) that allows for their efficient aminoacylation by tyrosyl-tRNA synthetase. This structural model rationalizes mutagenic and footprinting data that have established the importance of specific regions of the viral RNA for recognition by its replicase, (ATP,CTP):tRNA nucleotidyl-transferase and yeast tyrosyl-tRNA synthetase. The new fold has biological implications that can be used as a predictive tool for elaborating new experiments.
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PMID:Solution structure of the 3'-end of brome mosaic virus genomic RNAs. Conformational mimicry with canonical tRNAs. 828 79

Nuclear-encoded cytoplasmic tRNAs are imported into the mitochondria of kinetoplastid protozoa by an unknown mechanism. In a Leishmania in organello system, ATP-dependent import of a cloned, unspliced tRNATyr(GUA) transcript was demonstrated by protection from ribonuclease, whereas import of a tRNAGln(CUG) transcript was much less efficient. Specific binding of tRNATyr to two mitochondrial surface proteins of 15 and 22 kilodaltons was observed. Tubulin antisense-binding protein (TAB), the 15-kilodaton species, was purified to apparent homogeneity by RNA affinity chromatography. TAB forms stable complexes with the D stem-loop region of tRNATyr. Immunocytochemical and cell fractionation experiments, combined with limited proteolysis, suggested the association of TAB with the outer mitochondrial membrane. Importantly, anti-TAB antibody specifically inhibited binding as well as import of tRNATyr and of a synthetic structural homolog. These results support the role of TAB as a membrane-bound receptor or carrier for RNA import into Leishmania mitochondria.
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PMID:Role of an RNA-binding protein in import of tRNA into Leishmania mitochondria. 926 Nov 54

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade beta-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of beta-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).
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PMID:Aging, calorie restriction and ubiquitin-dependent proteolysis in the livers of Emory mice. 962 31

2',5'-Oligoadenylate [2-5(A)] synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5'-linked oligoadenylates in the presence of double-stranded RNA (dsRNA), their cofactor. The 2-5(A) molecules, in turn, activate the latent ribonuclease RNase L by promoting its dimerization. The 2-5(A) synthetase pathway has been implicated in interferon's antiviral and anticellular activities. In addition to their interesting cellular properties, these enzymes are also enzymologically interesting because they are the only known template and primer independent nucleotide (DNA or RNA)polymerases that synthesize 2'-5'-linked oligonucleotides. Moreover, their mode of activation by dsRNA remains unknown. In the past, biochemical and structure-function studies have been hampered by the lack of a convenient system for expressing recombinant 2-5(A) synthetases. These proteins are toxic to mammalian cells, probably because of RNase L activation, and proteins produced in bacteria do not have full enzymatic activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression of the small and medium isozymes. Here, methods are described for the production, purification, and characterization of the mouse small (9-2) (S. K. Ghosh, J. Kusari, S. K. Bandyopadhyay, H. Samanta, R. Kumar, and G. C. Sen, 1991, J. Biol. Chem. 266, 15293-15299) and human medium (P69) (I. Marie and A. G. Hovanessian, 1992, J. Biol. Chem. 267, 9933-9939) 2-5(A) synthetase isozymes and their mutants using the insect cell system. We also report methods for studying 2-5(A) synthetase-dsRNA interactions and protein-protein interactions among the subunits of the two isozymes.
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PMID:Production, purification, and characterization of recombinant 2', 5'-oligoadenylate synthetases. 973 8

RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis. One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases. Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L. Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p. In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L. To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue. The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities. The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize. These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.
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PMID:Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L. 986 63

Protein unfolding is a key step in the life cycle of many proteins, including certain proteins that are degraded by ATP-dependent proteases or translocated across membranes. The detailed mechanisms of these unfolding processes are not understood. Precursor proteins are unfolded and imported into mitochondria by a macromolecular machine that spans two membranes and contains at least nine different proteins. Here we examine import of a model precursor protein derived from the ribonuclease barnase and show that mitochondria unfold this protein by unraveling it from its N-terminus. Because barnase in free-solution unfolds by a different pathway, our results demonstrate that mitochondria catalyze unfolding in the way that enzymes catalyze reactions, namely by changing reaction pathways. The effectiveness of this mechanism depends on the structure of the N-terminal part of the precursor protein.
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PMID:Mitochondria unfold precursor proteins by unraveling them from their N-termini. 1058 40

It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. This study was performed to elucidate age-related changes in GH-dependent effects at the level of gene expression. Through the application of complementary DNA representational difference analysis (RDA) we have identified gene products that are reduced during aging in rat liver. The expression of these genes was restored upon GH treatment. Results from reverse Northern and ribonuclease protection analysis confirmed that the RDA products were truly differentially expressed. In addition to well characterized GH-regulated genes, including CYP2C12, CYP2C13, and alpha2u-globulin, we demonstrate the differential expression of at least 11 genes previously not known to be under GH control. Several hepatic transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were identified. Other RDA products consisted of transcripts encoding proteins involved in ATP synthesis, detoxification of reactive oxygen species, or immune responses. This list of GH-regulated genes in the old rat may shed further light on the action and mechanism behind the positive effects of GH on, for example, body composition and the immune system that have been observed in different animal and human studies.
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PMID:Differential cloning of growth hormone-regulated hepatic transcripts in the aged rat. 1069 65

Regulation of extracellular excitotoxins by glial and neuronal glutamate transporters is critical to maintain synaptic terminal integrity. Factors interfering with the normal functioning of these transporters might be involved in neurodegeneration. Among them, recent studies have shown that hypoxia alters glutamate transporter function; however, it is unclear if hypoxia has an effect on the expression of glutamate transporters and which intracellular signaling pathways are involved. The C6 rat glial and GT1--7 mouse neuronal cell lines were exposed to hypoxic conditions (5% CO(2), 95% N(2)) and levels of glutamate transporter mRNA were determined by ribonuclease protection assay. After 21 hr, there was a 100% increase in levels of rat excitatory amino acid transporter 3 (EAAT3) mRNA in C6 cells and a 600% increase in levels of murine EAAT2 mRNA in GT1--7 cells. There was a similar increase in mRNA levels after hypoxia in C6 cells transfected with human EAAT2, whereas reoxygenation normalized the expression levels of glutamate transporters. Although the expression of EAATs was associated with increased immunoreactivity by Western blot, functioning of the transporters was decreased as evidenced by D-aspartate uptake. Finally, although the protein kinase C stimulator phorbol-12-myristate-13-acetate enhanced EAAT2 mRNA levels after hypoxia, protein kinase C inhibitor bisindolylmaleimide I had the opposite effect. Taken together, this study suggests that the hypoxia is capable of upregulating levels of EAATs via a protein kinase C-dependent compensatory mechanism. This increased expression is not sufficient to overcome the decreased functioning of the EAATs associated with decreased ATP production and mitochondrial dysfunction.
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PMID:Altered expression of glutamate transporters under hypoxic conditions in vitro. 1128 47

2'-5' oligoadenylate (2-5 (A)) synthetases are major components of the antiviral pathways induced by interferons. In the presence of double-stranded RNA, they polymerize ATP to form 2-5 (A) oligomers that, in turn, activate the latent ribonuclease RNase L, causing mRNA degradation. These enzymes, unlike other nucleotidyl transferases, catalyze 2'-5', not 3'-5', phosphodiester bond formation between substrates bound to the acceptor and donor sites. Moreover, unlike other members of this extended family, the P69 isozyme of 2-5 (A) synthetase functions as a homodimer. Here, we report that the need for P69 dimerization is because of a crisscross enzyme reaction joining two substrate molecules bound to two opposite subunits. Consequently, although homodimers of mutants in the previously identified acceptor site, the donor site, or the catalytic site were inactive, selective heterodimers of the mutants were active because of subunit complementation. The catalytic site had to be present in the same subunit that contained the acceptor site, whereas the donor site had to be provided by the other subunit. These results allowed us to design a mutant protein that acted as a dominant-negative inhibitor of wt P69 but not of another isozyme of 2-5 (A) synthetase.
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PMID:Crisscross enzymatic reaction between the two molecules in the active dimeric P69 form of the 2'-5' oligodenylate synthetase. 1222 86


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