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At least 19 recordsLinked to original sources
PubMed1989

Exoribonuclease activity of purified reverse transcriptase preparations from retroviruses.

Highly purified and commercially available preparations of reverse transcriptases from retroviruses contain a 3' to 5' exoribonuclease activity capable of hydrolyzing synthetic homopolyribonucleotides having a 3'-OH end. The exoribonuclease activity of reverse transcriptase preparations from Rous associated virus-2 was further characterized. This exoribonuclease activity cleaves poly(C) and poly(U) exonucleolytically from the 3'-OH end to produce nucleoside 5'-phosphates. Poly(A), poly(G), circular polyribonucleotide, and double-stranded polyribonucleotide were not hydrolyzed by the activity. This is a novel type of exoribonuclease activity.

Buffers↗
PubMed1985

Simultaneous isolation of cytoplasmic endoribonuclease and exoribonuclease of Trypanosoma brucei.

An endoribonuclease and an exoribonuclease have been isolated simultaneously from the cytoplasm of Trypanosoma brucei by hydroxyapatite column chromatography. The endoribonuclease produced oligonucleotides from poly(adenylic acid) with 5'-phosphate and 3'-OH termini. The exoribonuclease produced only ribonucleoside 5'-phosphates from poly(adenylic acid). The relative rates of degradation of synthetic homopolynucleotides by the endoribonuclease under standard conditions were in the order poly(adenylic acid) greater than poly(uridylic acid) poly(cytidylic acid); for the exoribonuclease the order was poly(adenylic acid) poly(uridylic acid) greater than poly(cytidylic acid). Natural transfer and ribosomal RNAs were also degraded by both enzymes, while DNA was resistant to them. The optimal pH of activity for each enzyme was 7.5-8.0. Both ribonucleases require Ca2+ for maximum enzymatic activity.

Animals↗
PubMed1991

A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates.

We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component.

Adenosine Triphosphate↗
PubMed1992

The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli.

Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.

Bacterial Proteins↗
PubMed1984

Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli.

A single-strand-specific, nucleolar exoribonuclease from Ehrlich ascites tumor cells has been isolated and purified free from other nucleases. The exonuclease degraded single-stranded RNA processively from either a 5'-hydroxyl or a 5'-phosphorylated end and released 5'-mononucleotides. The enzyme digested single-strand poly(C), poly(U), and poly(A) equally well but did not degrade duplex poly(C).poly(I) or poly(A).poly(U). Less than 0.2% of duplex DNA or 1.5% of heat-denatured DNA was degraded under the conditions which resulted in greater than 26% degradation of RNA. The ribonuclease required Mg2+ (0.2 mM) for optimum activity and was inhibited by ethylenediaminetetraacetic acid but not by human placental RNase inhibitor. The native enzyme had a Stokes radius of 42 A and a sedimentation coefficient (S20,w) of 4.3 S. From these values, an apparent molecular weight of 76 000 was derived by using the Svedberg equation. The localization and unique mode of degradation suggest a role for the 5'----3' exoribonuclease in ribosomal RNA processing.

Animals↗
PubMed1991

Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1.

The portion of the internal transcribed spacer 1 found on 20S pre-rRNA accumulates in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1, showing that an endonucleolytic cleavage at the 3' terminus of 18S rRNA is involved in the 20S pre-rRNA to 18S mature rRNA conversion. Smaller fragments of the spacer sequence are also found. The exoribonuclease may be involved as a cytoplasmic RNase in the hydrolysis of the spacer.

Exoribonucleases↗
PubMed1991

mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo.

Two 3'-5' exoribonucleases, polynucleotide phosphorylase and ribonuclease II play a central role in the degradation of bacterial mRNA to ribonucleotides. Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3'-5' exoribonucleolytic attack in vivo by blocking the processive activities of these enzymes. For many mRNA species stem-loop structures appear to provide a very efficient block to decay from the 3' end, such that the rate-determining step for mRNA decay occurs elsewhere in the transcript. We have examined the stalling of 3'-5' exoribonucleases at stem-loop structures in vitro. Although stem-loop structures alone can impede the progress of both enzymes, the duration of stalling at these structures in vitro is insufficient to account for the increased half-lives that they confer on mRNA in vivo. These data suggest that an additional factor, such as a stem-loop binding protein, is required for stabilization of mRNA by stem-loop structures in vivo. The implications for the regulation of mRNA stability are discussed.

Amino Acid Sequence↗
PubMed1984

A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN.

A multiple mutant strain of Escherichia coli containing mutations affecting the exoribonucleases, RNase II, RNase D, and RNase BN, and also the endonuclease, RNase I, was constructed by P1-mediated transduction. Extracts of the mutant strain were lacking the aforementioned RNase activities. The multiple mutant displayed normal growth in both rich and minimal media at a variety of temperatures, recovered from starvation essentially as the wild-type parent, and could support the growth of a variety of bacteriophages. In addition, RNA synthesis was normal and no precursor RNA accumulation was observed. The properties of the mutant strain indicate that the three exoribonucleases are not essential for the viability of E. coli. The implications of these findings to our understanding of RNA processing and degradation are discussed.

Endoribonucleases↗
PubMed1985

Purification and mode of action of a microsomal exoribonuclease from rat liver.

An exoribonuclease has been purified nearly to homogeneity from rat liver microsomes and its mode of action and general properties were studied. The molecular weight values for the enzyme, as estimated by gel filtration and SDS-polyacrylamide gel electrophoresis, were 88 000 and 92 000, respectively. The enzyme produced, via a processive mechanism Ado5'P as the only product from poly(A). The results of the hydrolysis of 4 S (Ado5'P)n and (Ado3'P)n by the exoribonuclease with or without alkaline phosphatase and the inhibition of the enzymatic activity by oligonucleotides having a 3'-phosphate group in the 3'-terminus suggested that the degradation proceeds in the 3' to 5' direction. These findings were confirmed by the analysis of hydrolyzed products of various oligoadenylates and Ado3'PUrdPGuo and by the comparison of the rates of hydrolysis of (Ado3'P)2Ado by the enzyme in the presence of varying amounts of (Ado3'P)3. Mg2+ was required for the enzymatic activity, and Mn2+ partially substituted for Mg2+. The activity of the enzyme was stimulated by K+ and spermine.

Alkaline Phosphatase↗
PubMed1977

A kinetic analysis of the processive enzyme Lactobacillus plantarum exoribonuclease.

Oligonucleotide chains consisting of adenosine residues and ranging from 1 to 70 residues in length have been tested as substrates or inhibitors with Lactobacillus plantarum exoribonuclease (EC3.1.4.20). The kinetic constants V, Km, and Ki are all chain-length dependent. Ki decreases with increasing chain length to a minimum for oligonucleotides seven residues in length and then begins to increase slightly. Kinetic plots indicate that the oligonucleotides are almost all competitive inhibitors of poly A hydrolysis. However, the oligonucleotide (Ap)3A greater than p probably leads to mixed inhibition. The enzyme is unable to retain its processivity when it hydrolyzes short oligonucleotides such as (Ap)2A and (Ap)3A. It is proposed that L. plantarum exoribonuclease possesses seven binding sites for the polynucleotide. When the enzyme is bound to a long-chain-length substrate the complex is stabilized by a binding energy of about 8 Kcal/mol. After cleavage of the terminal nucleotide the remaining binding energy is still sufficient to maintain an enzyme-substrate complex. The shortened nucleotide chain is moved relative to the enzyme to re-form the seven-bond association by a gradient of energy of about 1.7 Kcal/mol for the change from six to seven bonds.

Adenosine↗
PubMedDate not supplied

[Isolation and some properties of exoribonuclease from the cell nuclei of the rat liver].

Exoribonuclease from rat liver cell nuclei was isolated and purified using selective extraction at pH 8.0, salting out with ammonium sulfate (30-50% saturation) and chromatography on DEAE-Sephadex A-50. The enzyme has the pH optimum 7.4-7.6, it requires Mg-2+. It catalyses the gradual removal of ribonucleoside-5'-monophosphates from synthetic polynucleotides and RNA; it preferably attacks single-stranded polynucleotides and is less efficient when hydrolysing poly-stranded helical polymers. It is supposed that exoribonuclease can participate in the destruction of non-informative regions in new-formed hyper-molecular RNA of cell nuclei.

Animals↗
PubMed1984

Influence of the xyloadenosine analogue of 2',5'-oligoriboadenylate on poly(A)-specific, 2',5'-oligoriboadenylate degrading 2',3'-exoribonuclease and further enzymes involved in poly(A)(+)mRNA metabolism.

The homogeneous poly(A)-specific 2',3'-exoribonuclease from calf thymus gland, which cleaves both 3',5'- and 2',5'-linked oligoriboadenylates, does not degrade (xyloA2'p)2 xyloA, the xylofuranosyladenosine analogue of the 2-5A core. This oligonucleotide, which is supposed to enter intact cells rapidly, was found to possess an increased stability and an enhanced antiherpesvirus activity compared to the natural (A2'p)2A (Eppstein, D.A., Barnett, J.W., Marsh, Y.V., Gosselin, G. and Imbach, J.-L. (1983) Nature 302, 723-724). The poly(A) anabolic enzyme, poly(A) polymerase (Mn2+-dependent), from the same source, which is initiated by (A3'p)2A and its higher oligomers, does not accept 2-5A core and its xyloadenosine analogue as primer. Both oligonucleotides exert no influence on endoribonuclease IV and on the integrity of the poly(A)-ribonucleoprotein complex.

Adenine Nucleotides↗
PubMed1987

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity.

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).

Exoribonucleases↗
PubMed1990

Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth.

As a step toward determining the metabolic role(s) of a 5'----3' exoribonuclease (XRN1), a yeast gene, XRN1, encoding XRN1, was first cloned, then disrupted to test its essentially or effect on yeast cell growth. Clones in the high-copy-number plasmid YEp24 cause overproduction (fivefold) of XRN1 in yeast cells, as measured by either poly(A) hydrolytic activity or immunoreactivity. Restriction mapping and deletion analysis showed that the XRN1 gene is located on a 6.7-kb XbaI-XhoI fragment of chromosome VII. The normal gene was disrupted in two haploid yeast strains by integrating a fragment with a BglII-deleted segment replaced with the yeast URA3 gene, and the disrupted strains lack XRN1. Successful transformation of haploid cells showed that the gene is not essential, but its absence markedly affected the cell growth rate. The growth defect is corrected by introduction of the XRN1 gene on a plasmid back into the disrupted yeast.

Blotting, Southern↗
PubMed1992

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.

Exoribonucleases↗
PubMed1987

A 5'----3' exoribonuclease of human placental nuclei: purification and substrate specificity.

An exoribonuclease that hydrolyzes single-stranded RNA by a 5'----3' mode yielding 5'-mononucleotides has been purified from human placental nuclei. Chromatographic studies of crude placental nuclear extracts suggest that the enzyme is a relatively abundant nuclear RNase. Poly(A) is degraded by a processive mechanism while rRNA is degraded in a partially non-processive manner, possibly because of its secondary structure. The enzyme has an apparent molecular weight of 113,000, derived from determinations of the Stokes radius (43 A) and sedimentation coefficient (6.3 S). Substrates with 5'-phosphomonoester end groups are 10-20 times better than 5'-dephosphorylated substrates. The locale of the enzyme in nuclei of normal human cells as well as its mode of action suggest a role in nuclear RNA processing or turnover.

Cell Nucleus↗
PubMed1991

Characterization of a novel NTP-dependent 3' exoribonuclease from yeast mitochondria.

We have purified and characterized a novel exoribonuclease that was isolated from the mitochondria of Saccharomyces cerevisiae. The enzyme degraded RNA in a 3' to 5' direction and was dependent on nucleotide triphosphates for activity. All eight of the standard ribo- and deoxyribonucleotide triphosphates supported activity with an apparent Km ranging from 20 to 90 uM. The enzyme also exhibited an RNA-dependent ATPase activity. Evidence suggests that in vivo the enzyme may associate with mitochondrial factors which can alleviate the dependence on nucleotide triphosphates for enzymatic activity. A model is discussed for the role of the enzyme in regulating the turnover of mitochondrial RNAs.

Adenosine Triphosphatases↗
PubMed1979

A microsomal exoribonuclease from rat liver.

A exoribonuclease has been purified from the microsomes of rat liver. The enzyme had an apparent molecular weight of 80 000-83 000 and produced, via a processive mechanism, 5'-AMP as the only product from poly(A). The degradation was found to proceed in the 3' to 5' direction. The relative rates of breakdown of synthetic polynucleotides by the enzyme under standard conditions were in the order poly(A) equal to poly(U) greater than poly(C). In addition to Mg2+, K+ was required for maximum activity. The enzymic activity was inhibited by p-chloromercuribenzoate and poly(G), but not by a rate liver RNAase inhibitor. The effect of spermine on the breakdown of synthetic polynucleotides by the enzyme has been studied. In the absence of K+, the breakdown of poly(C) was stimulated and that of poly(A) was stimulated slightly. However, the breakdown of poly(U) was inhibited slightly by spermine.

Animals↗