Adenylyl-(5′, 2′)-adenosine 5′-Phosphate ((2′-5′) pA-A) in Crude Crystals of 5′-Adenylic Acid Isolated from Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Adenylyl (5′,2′)-adenosine 5′-phosphate ((2′-5′)pA-A) was detected in crude crystals of 5′-AMP prepared from Penicillium nuclease (nuclease P₁) digest of a technical grade yeast RNA. While (3′–5′)A-A was split by nuclease P₁, spleen phosphodiesterase, snake venom phosphodiesterase or alkali, (2′–5′)A-A was not split by a usual level of nuclease P₁ or spleen phosphodiesterase. Nuclease P₁ digests of 12 preparations of technical grade yeast RNA tested were confirmed to contain (2′–5′)pA-A. Its content was about 1 to 2% of the AMP component of each RNA preparation. As poly(A) was degraded completely by the Penicillium enzyme into 5′-AMP without formation of any appreciable amount of (2′–5′)pA-A, the technical grade RNA is supposed to contain 2–5′ phosphodiester linkages in addition to 3′–5′ major linkages.     

A positive zymogram method for ribonuclease

We have developed a rapid, sensitive, and specific zymogram for detecting ribonuclease (RNase). The method makes use of an agarose gel containing the small substrate UpA [uridylyl (3′ → 5′)-adenosine]. UpA is hydrolyzed by RNase to adenosine, which is deaminated by adenosine deaminase. The inosine so formed is linked by a series of enzymatic reactions (nucleoside phosphorylase, xanthine oxidase) to formation of a blue tetrazolium salt. This method is superior in that it entails a staining reaction only at sites of RNase activity (positive zymogram) rather than clearing of a background of RNA (negative zymogram), a process which is often mimicked by protein devoid of RNase activity.     

Conformational Flexibility of Two RNA Trimers Explored by Computational Tools and Database Search

Abstract

Two RNA sequences, AAA and AUG, were studied by the conformational search program CICADA and by molecular dynamics (MD) in the framework of the AMBER force field, and also via thorough PDB database search. CICADA was used to provide detailed information about conformers and conformational interconversions on the energy surfaces of the above molecules. Several conformational families were found for both sequences. Analysis of the results shows differences, especially between the energy of the single families, and also in flexibility and concerted conformational movement. Therefore, several MD trajectories (altogether 16 ns) were run to obtain more details about both the stability of conformers belonging to different conformational families and about the dynamics of the two systems. Results show that the trajectories strongly depend on the starting structure. When the MD start from the global minimum found by CICADA, they provide a stable run, while MD starting from another conformational family generates a trajectory where several different conformational families are visited. The results obtained by theoretical methods are compared with the thorough database search data. It is concluded that all except for the highest energy conformational families found in theoretical result also appear in experimental data.

Registry numbers:

adenylyl-(3′ →5′)-adenylyl-(3′ →5′)-adenosine [917-44-2]

adenylyl-(3′ →5′)-uridylyl-(3′ →5′)-guanosine [3494-35-7]     

(2′–5′)An-dependent RNase: Enzyme levels are decreased in barrier-reared control and IFN-β or IFN-γ treated Balb/c mice

(2′–5′)A_n-dependent RNase functions as a translational regulatory protein which mediates interferon action. Levels of this enzyme are decreased in barrier-reared Balb/c (+/+), Balb/c (+/nu), and Balb/c (nu/nu) mice when compared to conventionally reared Balb/c (+/+) mice. This suggests that high levels of (2′–5′)A_n-dependent RNase in conventionally reared mice are maintained by continuous exposure to microbial flora which may induce interferons. Interferon treatment of barrier-reared mice does not, however, result in an increase in (2′–5′)A_n-dependent RNase levels. This suggests that responsiveness to interferons is decreased in barrier-reared mice. The high levels of (2′–5′)A_n-dependent RNase which are maintained in normal mice under physiological conditions may be important for rapid and effective defense against viral pathogens.     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

Crystal structure of the C‐terminal 2′,5′‐phosphodiesterase domain of group a rotavirus protein VP3

In response to viral infections, the mammalian innate immune system induces the production of the second messenger 2′–5′ oligoadenylate (2–5A) to activate latent ribonuclease L (RNase L) that restricts viral replication and promotes apoptosis. A subset of rotaviruses and coronaviruses encode 2′,5′‐phosphodiesterase enzymes that hydrolyze 2–5A, thereby inhibiting RNase L activation. We report the crystal structure of the 2′,5′‐phosphodiesterase domain of group A rotavirus protein VP3 at 1.39 Å resolution. The structure exhibits a 2H phosphoesterase fold and reveals conserved active site residues, providing insights into the mechanism of 2–5A degradation in viral evasion of host innate immunity. Proteins 2015; 83:997–1002. © 2015 Wiley Periodicals, Inc.     

Synthesis and RNase L Binding and Activation of a 2-5A-(5′)-DNA-(3′)-PNA Chimera,a Novel Potential Antisense Molecule

Abstract

Fully automated solid-phase synthesis gave access to a hybrid in which 5′-phosphorylated-2′-5′-linked oligoadenylate (2–5A) is connected to the 5′-terminus of DNA which, in turn, is linked at the 3′-end to PNA [2–5A-(5′)-DNA-(3′)-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.     

Neuraminidase deficiency in the cherry red spot-myoclonus syndrome

Two patients with the cherry red spot-myoclonus syndrome excreted excessive quantities of sialylated oligosaccharides in urine. Skin fibroblasts from both patients had a severe deficiency of neuraminidase activity using α-L-N-acetylneuraminosyl-(2→6′) lactose and a sialylhexasaccharide from their urine as substrates. A less severe deficiency was found using 2-(3′ methoxyphenyl)-N-acetylneuraminic acid, fetuin, and α-L-N-acetylneuraminosyl-(2→3′) lactose as substrates. I propose that the primary enzyme defect in this autosomal recessive disorder is a deficiency of lysosomal neuraminidase.     

Purification and characterization of the Escherichia coli exoribonuclease RNase R. Comparison with RNase II

Escherichia coli RNase R, a 3' --> 5' exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of approximately 95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mm Mg(2+) and 50-500 mm KCl. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5'-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3'-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3'-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.     

The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures.

Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method.     

Sse9I restriction-modification system: Organization of genes and structural comparison of proteins

The nucleotide sequence was established for the operon of the Sse9I type II restriction-modification system of Sporosarcina species 9D. The enzymes of the Sse9I system recognize the 5′-AATT-3′ tetranucleotide. The operon includes three genes, sse9IC-sse9IR-sse9IM, which are transcribed unidirectionally and code, respectively, for the controller protein (C.Sse9I), restriction endonuclease (R.Sse9I), and DNA methyltransferase (M.Sse9I). The region immediately upstream of sse9IC was found to contain a conserved nucleotide sequence (C box) providing a binding site for C. Sse9I. The amino acid sequences of C.Sse9I and R.Sse9I were compared with those of related proteins. In the case of R.Sse9I, the highest homology was observed with the R.MunI (5′-CAATTG-3′) and R.EcoRI (5′-GAATTC-3′) regions that harbor the amino acid residues involved in recognizing the AATT inner tetranucleotide. The sse9IR gene was cloned in an expression vector, and recombinant R.Sse 9I was isolated.     

A proton magnetic resonance study of the interaction of adenylyl (3' is greater than 5') adenosine with polyuridylic acid

The interaction of adenylyl (3′ → 5′) adenosine (ApA) with polyuridylic acid in D₂O solution at neutral pD has been studied by high resolution proton magnetic, resonance spectroscopy. At temperatures above ～32°C, no evidence was obtained for the interaction of ApA with poly U. Below this temperature, a rigid triple-stranded complex involving a stoichiometry of 1 adenine to 2 uracil bases is formed, presumably via specific adenine–uracil base-pairing and cooperative base stacking of the adenine bases in a manner similar to that previously reported for the adenosine–poly U complex.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Aspergillus quercinus (IFO 4363) was selected as the most suitable strain to produce 5′-mononucleotides from RNA among several species of Aspergillus which produced enzymes capable of degrading RNA into 5′-mononucleotides.

Aspergillus quercinus produced two kinds of RNA-depolymerases (designated as RNA-deploymerase I and II), phosphodiesterase, phosphomonoesterase and adenylic deaminase in the culture medium. The optimum pH of each enzyme was found to be about 4.5, 7.0, 5.0, 6.0 and 5.5, respectively. Maximal production of these enzymes in the culture medium occurred at 96, 96, 216, 168 and 264 hour culture, respectively. The culture filtrate of Aspergillus quercinus degraded RNA into 3′-mononucleotides at the pH lower than 6.0, into 5′-mono-nucleotides at the pH higher than 8.5 and into both mononucleotides at the pH range between 6.0 and 8.5. 5′-Inosinic acid was prepared from RNA by using the extra- and intracellular enzymes of Aspergillus quercinus.     

Partial purification and characterization of the components of the neutral ribonuclease II-inhibitor system of normal and distrophic mouse skeletal muscle

The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.127.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 degrees C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 degrees C. The active RNase II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH-activity profiles in 200 mM KC1 with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KC1 concentration is increased above 50 mM KC1. RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 degrees C in 5 min. These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II - inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II - inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.     

The signal transfer regions of G alpha(s)

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.     

The mode of action of divalent cations on the RNase catalyzed hydrolysis of RNA and uridylyl (3'-5') uridine

Pseudo first order rate constants (k′) have been measured for the RNase A catalyzed hydrolysis of uridylyl (3′–5′) uridine at several ionic strengths and compositions. The k′ values are independent of Mg²⁺ concentration between 0 and 10 mM. This shows that for hydrolysis of RNA, in which Mg²⁺ concentration does change k′, the perturbation must be through binding of Mg²⁺ to the substrate RNA rather than to the enzyme RNase.