Endoribonuclease from bovine adrenal cortex cytosol.

An endoribonuclease which digests a variety of synthetic homoribopolymers and poly(A)-rich mRNA has been identified and purified greater than 500-fold with respect to specific activity from bovine adrenal cortex cytosol. Enzymatic digestion of synthetic poly(riboadenylic acid) was stimulated by Mn-2+ and Mg-2+ and the enzyme exhibited broad pH and salt optima. Poly(cytidylic acid) and poly(uridylic acid), but not poly(guanylic acid), served as substrates for the enzyme preparation; double-stranded RNA, DNA, and DNA-RNA hybrids were not digested by the enzyme. Digestion generated oligonucleotides with 3-hydroxyl and 5'-monophosphoester termini. On isoelectric focusing, the enzymatic activity banded at pH 8.3 plus or minus 0.2. An initial preferential cleavage of the poly(A) tract of poly(A)-rich RNA is suggested by the rapid appearance of a 4-6S digestion product highly enriched for adenylic acid; however, progressive digestion of the RNA occurs with additional incubation.     

Extending the limits to enzymatic catalysis: diffusion of ribonuclease A in one dimension

Bovine pancreatic ribonuclease A (RNase A) is a distributive endoribonuclease that catalyzes the cleavage of the P-O5' bond of RNA on the 3' side of pyrimidine residues. Here, RNase A is shown to cleave the P-O5' bond of a pyrimidine ribonucleotide faster when the substrate is embedded within a longer tract of poly(adenylic acid) [poly(A)] or poly(deoxyadenylic acid) [poly(dA)]. These data indicate that a ribonuclease can diffuse in one dimension along a single-stranded nucleic acid. This facilitated diffusion is mediated by Coulombic interactions, as the extent is diminished by the addition of NaCl. RNase A is more effective at cleaving a pyrimidine ribonucleotide embedded within a poly(dA) tract than within a poly(deoxycytidylic acid) [poly(dC)] tract. T45G RNase A, which catalyzes the processive cleavage of poly(A) but the distributive cleavage of poly(cytidylic acid) [poly(C)], has the same preference. Apparently, processive catalysis by the T45G enzyme arises from the expanded substrate specificity of the variant superimposed upon an intrinsic ability to diffuse along poly(A). Homologous ribonucleases with cytotoxic activity may rely on facilitated diffusion along poly(A) tails for efficient degradation of the essential information encoded by cellular mRNA.     

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.     

Purification and properties of a ribonuclease from corn leaf tissues

An acid endoribonuclease isolated from corn leaf tissues was purified 530 times. Gel electrophoresis indicated that the enzyme was homogeneous. The enzyme showed an optimum pH at 5.5 and an apparent molecular weight of 32 000. Corn RNase attacks natural RNAs and synthetic polyribonucleotides and the relative rate of degradation was poly U > yeast RNA > E. coli tRNA > poly A ⪢ poly C. Zn²⁺, Mg²⁺, Mn²⁺ and EDTA inhibited the enzyme activity. No stimulation by K⁺ was observed. Cu²⁺ and heparin had no effect on the activity. The results suggest that the investigated RNase differs from other known corn ribonucleases.     

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.     

5S RNA may not have highly ordered structure like 4S RNAS

The rates of hydrolysis of the following polyribonucleotides as catalysed by RNase I, an enzyme specific for single stranded RNAs, follow the sequence shown; poly (A) > 23S RNA > 5S RNA ? 16S RNA > 4S RNA = poly (I). poly (C). The rates were measured by direct spectrophotometric as well as by trichloroacetic acid precipitation methods. The extents of inhibition of RNase I-catalysed hydrolysis of poly (A) by each of the above-mentioned polyribonucleotides follow the reverse order. Taking into account the fact that double stranded RNAs are inhibitory to RNase I it may be concluded from the above results that 5S RNA has much less ordered structure than 4S RNAs. This prediction is contrary to expectations and its validity will be known when the tertiary structure of 5S RNA will be worked out. These results also indicate that 16S RNA may have more folded structure than 23S RNA.     

Interaction of eukaryotic tyrosyl-tRNA-synthetase with high molecular weight RNA

The affinity of eukaryotic tyrosyl-tRNA synthetases from bovine liver and from yeast for E. coli ribosomal RNA and synthetic polyribonucleotides has been studied by protein binding on the rRNA-Sepharose column and enzyme inhibition by high molecular weight RNAs. Tyrosyl-tRNA synthetase from bovine liver (Mr 2.59 kDa) was fully retained on the rRNA-Sepharose and eluted by buffer with 100 mM KCl. The functionally active modified form of bovine liver tyrosyl-tRNA synthetase obtained by endogenous limited proteolysis (Mr 2.38 kDa) partially maintains the affinity for rRNA and is eluted by 50 mM KCl. The highest rRNA-binding ability was revealed for yeast tyrosyl-tRNA synthetase eluted by 200 mM KCl. The E. coli tyrosyl-tRNA synthetase was not retained on rRNA-Sepharose. The aminoacylation activities of both bovine liver and yeast tyrosyl-tRNA synthetases were efficiently inhibited by rRNA and the inhibition was partially competitive in respect to tRNA(Tyr). At the same time the activities of proteolytically modified bovine tyrosyl-tRNA synthetase and E. coli tyrosyl-tRNA synthetase were not influenced by the addition of rRNA. Synthetic single- and double-stranded polyribonucleotides specifically inhibited the activity of bovine tyrosyl-tRNA synthetase to different extent. The inhibition degree of bovine liver tyrosyl-tRNA synthetase decreased in the order: poly (G) greater than poly (I) greater than poly (I).poly (C) greater than poly (G).poly (C) greater than poly (C) greater than poly (A). Poly (U) did not inhibit the activity of bovine liver tyrosyl-tRNA synthetase.     

Poly(adenylic acid) in small amounts, free or covalently linked to substrate, protects RNA from hydrolysis by ribonuclease.

Short lengths (18 residues) of poly(A), covalently linked to the 3'-termini of Escherichia coli 5 S rRNA, induce powerful inhibitions (38-87%) of the activities of RNAases (ribonucleases) from Citrobacter sp., Enterobacter sp., bovine pancreas, human spleen and human plasma. As the polypurine chain length is extended, enzyme activity declines. Furthermore, poly(A) sequences, present only on a small subpopulation of RNA, and accounting for less than 1% of total RNA, serve to protect all RNA, polyadenylated or not, from enzyme-catalysed degradation. The quantity of 3'-terminal adenylic acid residues, relative to the amount of substrate, determines enzyme activity. The exact distribution of a fixed amount of poly(A) residues on the 3'-termini of substrate molecules is unimportant in this respect. Comparison of the efficacies of inhibition of RNAase activity, by using linked poly(A) and similar quantities of free poly(A), revealed that although the free polypurine inhibits RNAase activity, covalent linkage of poly(A) to RNA is more advantageous to the stability of an RNA substrate. However, the ratio of inhibited activities obtained by using linked or free poly(A) may change considerably with alterations in either substrate concentration or polyadenylic acid segment length.     

Nuclease Stn α from <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis>: Characterization of the Associated Adenylic Acid Preferential Ribonuclease Activity

Nuclease Stn α from Streptomyces thermonitrificans hydrolyses DNA and RNA at the rate of approximately 10:l. The optimum pH and temperature for RNA hydrolysis were 7.0 and 45°C. The RNase activity of nuclease Stn α had neither an obligate requirement of metal ions nor was it activated in the presence of metal ions. The enzyme was inhibited by Zn²⁺, Mg²⁺, Co²⁺, and Ca²⁺; inorganic phosphate; pyrophosphate; NaCl; KCl; and metal chelators. It was stable at high concentrations of urea but susceptible to low concentrations of Sodium dodecyl sulfate and guanidine hydrochloride. The rates by which nuclease Stn α hydrolysed polyribonucleotides occurs in the order of poly A >> RNA >> poly U > poly G > poly C. The enzyme cleaved RNA to 3′ mononucleotides with preferential liberation of 3′AMP, indicating it to be an adenylic acid preferential endonuclease.     

Isolation and Characterization of a Relatively Guanine-specific Endoribonuclease in Rice Bran

A relatively guanine-specific endoribonuclease (RB-1) was isolated from rice bran. The pH optimum was 8.5 using yeast RNA as a substrate. The enzyme activity was inhibited by Cu²⁺, Zn²⁺, DTT and SDS, while EDTA, PCMB, IAA and heparin had no effect on the activity. The enzymic activity of RB-1 was inhibited by 3′-GMP as an end-product inhibitor. The enzyme protein was highly heat-stable. RB-1 did not hydrolyze native calf thymus DNA, heat-denatured DNA, poly A, poly U and poly C. Among synthetic substrates, only poly I was depolymerized. Only 2′,3′-cyclic GMP was identified in the hydrolysate of yeast RNA after 6hr hydrolysis.     

Messenger ribonucleic acid metabolism in mammalian mitochondria: relationship between the decay of mitochondrial mRNA and their poly(A).

The mitochondrial poly(adenylic acid)-containing mRNAs in mouse ascites cells pulsed with radioactive precursors contain 35-55 nucleotide long poly(adenylic acid) sequences. These sequences are shortened with age in cells chased with cold medium. The possible relationship between the decay rates of mRNA and their poly(adenylic acid) sequences has been investigated by using mitochondrial RNA and protein synthesis inhibitors. The pattern of mRNA decay as determined by a new solid-phase-bound complementary DNA procedure indicates the presence of two classes of poly(adenylic acid) containing mRNA in mammalian mitochondria: one decaying with a t1/2 of 45 min and the other class with a t1/2 of 210 min. Inhibitors such as ethidium bromide and puromycin which accelerate the decay of mitochondrial mRNA also cause an enhanced decay of poly(adenylic acid) sequences. These results have been interpreted as evidence supporting the involvement of poly(adenylic acid) sequences in the mRNA stability.     

Purification and some properties of a nuclease from rye germ nuclei

1. An endonuclease has been isolated from the nuclei of rye (Secale cereale L) germ and partially purified. The enzyme shows optimum activity over the pH range 5.4-7.4 towards both DNA and RNA, and has no phosphomonoesterase or phosphodiesterase activity. 2. DNA is degraded by the rye germ nuclease to oligonucleotides of similar size, and RNA to oligonucleotides and mononucleotides containing a C-terminal 5'-phosphate group. 3. The rate of hydrolysis of nuclear acids by the enzyme decreases in the following order: native DNA greater than denatured DNA greater than RNA. Synthetic polynucleotides are hydrolysed at a rate decreasing in the order: poly(A) greater than poly(U) greater than poly(C) greater than poly(G).     

A yeast endoribonuclease stimulated by Novikoff hepatoma small nuclear RNAs U1 and U2

Using [³H]m⁷Gppp[¹⁴C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of $\text{Saccharomyces}\text{cerevisiae}$. The [³H]m⁷Gppp[¹⁴C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [³H]m⁷Gppp[¹⁴C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA.     

Virus-Associated Nucleases: Location and Properties of Deoxyribonucleases and Ribonucleases in Purified Frog Virus 3

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (pH 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (pH 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA], pH 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (pH 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6S. Qbeta 6S DS RNA and 4S transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.     

Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.     

Purification and characterization of an endoribonuclease from nucleoplasm and nucleoli of HeLa cells.

An endoribonuclease has been isolated from HeLa cell nuclei. Approximately 70% of the enzyme appears to be nucleolar bound; 30% is in the nucleoplasm. Studies of the purified enzyme reveal that the enzyme is an endonuclease of estimated molecular weight 16,000. It produces oligonucleotides bearing 5'-phosphate end groups. The enzyme degrades poly(C) and poly(U), as well as rRNA and heterogeneous nuclear RNA, Poly(A), double-stranded RNA, and DNA are not cleaved. The enzyme is heat-labile and is inhibited by 10mM Mg2+ and 50 mM NaCl. The enzyme is probably distinct from previously described nuclear endonucleases.