Terminal Nucleoside Research Articles

Interaction between RNA-Dependent RNA Polymerase of Alfalfa Mosaic Virus and Its Template: Oxidation of Vicinal Hydroxyl Groups Blocksin VitroRNA Synthesis

In the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependentin vitroRNA synthesizing system of alfalfa mosaic virus (AlMV) we were able to study the effect of small chemical modifications of the 3′ end of the template RNAs on product formation. After oxidation of the 3′-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuylet al., Virology176, 346–354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3′ end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AlMV displays a high specificity for its cognate template, either before or after the oxidation of the 3′-terminal nucleoside. From this it was concluded that periodate modification of the 3′-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadtet al., Virology182, 309–315, 1991), was not the main target involved in the inhibition of RNA synthesis.

Conformational studies of thymidine dimers containing sulfonate and sulfonamide linkages by NMR spectroscopy

The conformations of 3′-azido-terminated-sulfonate-dimer 1 and 3′-amino-terminated-sulfonamide-dimer 2 are characterized by the following features: (1) The 5′-terminal nucleoside moiety of 1 has a S-type sugar (87% S), a staggered γ + (rotamer across the C4′C5′ bond (65%) and an anti orientation of the base about the glycosidic bond. The 5′-terminal nucleoside moiety of 2 has an almost equal population of S and N conformations, a staggered γ + rotamer (69 %) and an anti orientation of the base. (2) The 3′-terminal nucleoside moieties of 1 and 2 are in ∼50% N ⇄ S equilibrium and the γ t conformer is the most populated. A comparison of the conformational properties of 1 and 2 with the natural thymidyl(3′ → 5′)thymidine [d(TpT)] 3, thymidylyl-(3′ → 5′)-5′-thio-5′-deoxythymide d(TpST) 5 4 and thymidinylacetamido-[3′(O)→5′(C)]-5′-deoxythymidine NH 2d(TcmT) 5 5, show the following characteristics: (i) The conformational preference of the sugar ring is partially determined by the gauche effect. This means that the more polar the C3′X bond due to the electronegative character of the 3′-α-X substituent, the more than N ⇌ S equilibrium is biased toward the S-type conformation: 3′-O-S > 3′-O-H > 3′-N 3 > 3′-NH 2. (ii) The conformation about the C4′C5′ bond (γ) is also influenced by the gauche effect based on the nature of the 5′-substituent and by the ability of the 5′-substituent to form hydrogen bonding with the H6 of thymine. Thus, the population of the γ + conformer follows the order: 5′-O > 5′-N > 5′-S > 5′-C. (iii) The C5′C6′ bond has a slight preference for the β t conformation (56 % β t in 1 and 58 % β t in 2), while in natural d(TpT) 3, the C5′O5′ bond accounts for 83% of β t conformation. Upon substitution of the 5′-oxygen by 5′-sulfur, as in d(TpST) 4, the population of β t conformer was found to decrease to 57 %. This decrease in the β t population in 1, 2 and 4 is a result of the reduced polarity of the 5′CX [X = CH 2 in 1 and 2 and X  S in 4] in comparaison to the 5′CO bond in 3, which weaken the gauche effect.

Dinucleoside Phosphates Containing Arabinose or Deoxyxylose. Hydrolysis by Exonucleases and Stacking Properties

Abstract Two pairs of isomeric dinucleoside monophosphates containing either 1-β-D-arabinofuranosyl)uracil (arabino-U, aU) or 1-β3-D-2-deoxy-threo-pentofuranosyl) thymine (2′-deoxyxylo-T, dxT) were synthesized. The kinetics of the hydrolysis of these dimers by venom and spleen phosphodiesterases (PDE) were studied. In addition, circular dichroism was used to study their stacking behaviour. A correlation was found to exist between the enzymatic cleavage rate and the conformation of the dimers. It was found that the configuration change at C-2′ or C-3′ in the 5′-terminal nucleoside residue, especially the inversion of 3′-hydroxyl involved into the internucleotide 3′-5′-phosphodiester linkage formation, was critical.

A route to 2′,5′-oligoadenylates with increased stability towards phosphodiesterases

The rates of enzymatic hydrolysis of 2′,5′-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3′-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2′-5′)adenylyl(2′-5′)adenosine.

Präparative darstellung von homologen der oligoribouridylsäure durch selektive partialhydrolyse von rna

Preparative preparation of homologues of oligoribouridylic acid by selective partial hydrolysis of RNA The preparation of oligouridylic acids was achieved by the stepwise partial hydrolysis of RNA using enzymatical and chemical methods of degradation followed by chromatographic purification steps. After RNA is submitted after first to RNAse T 1 and after that to RNase U 2 it is cleaved into oligopyrimidine nucleotides carrying purine nucleotides only at their 3′-terminal. By chemical deamination the pyrimidine sequences are converted to oligouridine sequences. After enzymatical dephosphorylation of the oligouridylic acids their 3′-terminal purine nucleosides are eliminated by a periodate-lysine treatment. The phosphate groups remaining at the 3′-terminal of the resulting oligouridylic acids are enzymatically removed. As a result of the partial hydrolysis the mean recoveries of the partial homologues are U 2-U 7 93% and U 8 86% adding up to about 0.5% of the amount of RNA used as starting material.

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.

Solid-phase synthesis of protected oligonucleotide blocks : Applications to block condensation on a polymer support and synthesis of a 3'-modified oligonucleotides

The solid-phase synthesis of protected oligodeoxy-ribonucleotide blocks using a new phosphoramidate linkage between the 3'-terminal nucleoside and the polymer support is described. The oligonucleotide block cleaved from the support contains a phosphodiester group at its 3'-terminus and can be used as a coupling unit in the solid-phase synthesis of long-chain oligonucleotides. A 35mer has been synthesized using hepta- and nonanucleotide blocks. By deprotection of the appropriate oligonucleotide block, a 16mer containing an o -chlorophenyl phosphate group at the 3'-terminus has been obtained.

Mechanism and base specificity of DNA breakage in intact cells by neocarzinostatin

When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of Terminal Nucleoside Phosphates and Thiophosphates via Phosphoramidite Chemistry

Abstract Synthetic oligonucleotides for use in biological processes often require a 5′-terminal phosphate function. To study enzymatic reactions this group can be replaced by a thiophosphate function. Most chemical methods for the synthesis of these termini base on phosphotriester chemistry.

Deoxyribonucleic acid damage by neocarzinostatin chromophore: strand breaks generated by selective oxidation of C-5' of deoxyribose.

Among the lesions induced in DNA by neocarzinostatin chromophore are spontaneous and alkali-dependent base release, sugar damage, and single-strand breaks with phosphate (PO4) at their 3' ends and PO4 or nucleoside 5'-aldehyde at the 5' ends. By measuring alkali-dependent thymine release and decomposition of the 5'-terminal thymidine 5'-aldehyde in drug-cut DNA, we show that the kinetics are the same for each process and that the nucleoside aldehyde is the source of about 85% of alkali-dependent thymine release. Reduction of the 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase permits their selective quantitation. Nucleoside 5'-aldehyde so measured accounts for over 80% of the drug-generated 5' ends; the remainder have PO4 termini. Since these techniques also include the contribution of alkali-labile sites in the measurement of PO4 ends, DNA sequencing was used to measure the ends directly. Using 3'-32P end-labeled DNA restriction fragments as substrates for the drug, it was found that drug attack at a T results in mainly two bands--the stronger one represents oligonucleotide with 5'-terminal nucleoside 5'-aldehyde and may account for over 90% of a particular break. Its structure was verified by its isolation from the sequencing gel, followed by various chemical and enzymatic treatments. In each case, the mobility of the product on the gel was altered in a predictable manner. In addition to spontaneous breaks, neocarzinostatin also causes alkali-labile breaks preferentially at T residues. These sites are heterogeneous in their sensitivity to alkali and are protected by reduction.

Tandem promoters in the gene for ribosomal protein S20.

Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G. A. (1981) J. Biol. Chem. 256, 8177-8182). We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro. In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts. The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate. In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence. A minor start occurs at residue 142 and uses CTP as the initiating nucleotide. In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively. In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder. Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation. Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.

Blocked and methylated 5'-terminal cap structures of rat brain messenger ribonucleic acids.

The presence and identity of 5'-terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [3H]sodium borohydride reduction or by beta-elimination of 5'-terminal nucleoside and incorporation of 32P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7-methylguanosine (m7G) as the 5'-terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5'-penultimate position was 6-methyl-2'-O-methyladenosine [m6A(m)] followed by 2'-O-methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2'-O-Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2'-O-methylcytidine [C(m)] and 2'-O-methyluridine [U(m)].

Conformational flexibility in single-stranded oligonucleotides: crystal structure of a hydrated calcium salt of adenylyl-(3'--5')-adenosine.

The crystal and molecular structure of a hydrated calcium salt of adenylyl-(3'--5')-adenosine(ApA) was determined from X-ray diffraction data collected on an automated diffractometer. Crystals of the salt are orthorhombic, space group P21212, with a = 30.614 (3), b = 17.894 (2), and c = 5.373 (1) A. The structure was solved by a combination of Patterson and direct methods and refined by least squares. The final value of the R index is 0.08. The 5'-terminal adenosine residue has a C(2')-endo ribose and assumes a syn conformation, which is stabilized by an O(5')-H...N(3) hydrogen bond within the nucleoside. The 3'-terminal nucleoside has a C(3')-endo ribose and is in the anti conformation. Both omega and omega', the torsion angles within the phosphodiester group, are approximately 60 degrees. Adenine bases from adjacent anions are joined by pairs of N(6)-H...N(1) hydrogen bonds and are stacked with symmetry-related bases. The calcium ion is bound to the dinucleoside phosphate by a direct interaction with the phosphate group and by outer-sphere, ligand-mediated interactions with O(2') of the 5'-terminal nucleoside and N(7) of the 3'-terminal nucleoside. This tridentate interaction of the ApA anion with the calcium coordination sphere probably enhances the stability of the observed ApA conformation. When combined with other crystallographic studies of ApA conformations, the crystal structure of this calcium salt provides additional evidence that dinucleoside phosphates have considerable conformational flexibility.

Process of cap formation of messenger RNA by vaccinia virus particles carrying an organized enzymes system.

Vaccinia virus mRNAs carry the cap structure m7G5'pppAm- or m7G5'pppGm- at the 5'-terminus, which is synthesized by a series of RNA polymerase and capping enzymes contained in the virus particle. The process of the cap formation at the 5'-terminus of mRNA was studied using an in vitro system under similar conditions to those of vaccinia virus multiplication in its host cell. After adding a methyl-group donor, methyl-3H-S-adenosylmethionine, the oligonucleotides, which were the de novo synthesized 5'-terminal part of mRNA, were isolated from the RNA-synthesizing virion at appropriate time intervals, and were analysed. The 5'-5' confronting nucleotides with 2'-O-methylation, G5'pppAm and G5'pppGm, were found with the completed cap structure, m7G5'pppAm and m7G5'pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m7G5'pppA and m7G5'pppG, were not detected at any incubation time, and it was concluded that methylation at the 2'-position of the 5'-terminal purine nucleoside of mRNA precedes methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated from vaccinia virus (Moss et al., 1976) and also from the results obtained using other kinds of virus particles, which carry RNA polymerase and capping enzymes. These differences may be due to the specific organization of a series of capping enzymes and RNA polymerase in each virus particle.

Synthesis of dicytidylyl-(3'-5')-1,2-di(adenosin-N6-yl)ethane and dicytidylyl-(3'-5')-1,4-di(adenosin-N6-yl)butane: covalently joined terminals of two transfer ribonucleic acids and their behavior toward snake venom phosphodiesterase.

The chemical synthesis of the tital bridged trinucleoside diphosphates 3e and 3f along with the corresponding dinucleoside phosphates 3c and 3d is described. Bridged nucleosides 3a and 3b gave on treatment with triethyl orthoformate in the presence of p-toluenesulfonic acid in dimethylformamide the cyclic orthoesters 2a and 2b. Condensation of 2a and 2b with N,2',5'-O-triacetylcytidine 3'-phosphate (1) using dicyclohexylcarbodiimide in pyridine afforded after deblocking and chromatographic separation products 3c-f. The latter were readily degraded with pancreatic RNase, but 3c and 3e were completely resistant toward snake venom phosphodiesterase whereas 3d and 3f were digested to the extent of 65 and 43%, respectively. The major product of degradation of 3f with phosphodiesterase was compound 3d resulting from the combined action of phosphodiesterase and contaminating phosphomonoesterase. The results are explained in terms of stacking of terminal bridge nucleoside units in 3c-f. The implications of these findings for the function of snake venom phosphodiesterase are discussed.

Characterization of a ribonuclease from Anacystis nidulans infected with cyanophage as-1

In Anacystis nidulans the ribonuclease (RNase) activity is very low but is greatly increased upon phage-infection. A RNase was isolated and purified over 300-fold from A. nidulans cells infected by cyanophage AS-1. The enzyme did not attack single- or double-stranded DNA, was inactive on p-nitrophenyl phosphate or bis- p-nitrophenyl phosphate as substrates, and had neither 3′- nor 5′-nucleotidase activity. The approximate MW of the enzyme was 12000. Maximal enzyme activity was at pH 7.5. No absolute requirement for metal ions was observed, but Fe 3+ stimulated and Co 2+ and Ni 2+ inhibited enzyme activity. The enzyme is an endonuclease which, upon exhaustive hydrolysis, produces mainly oligonucleotides (average chain-length: 3) with 3′-P termini. Analysis of the base composition of these oligonucleotides and determination of their 3′-terminal nucleosides, together with the investigation of the rate of hydrolysis of synthetic polyribonucleotides, have shown that the enzyme has a relative specificity for uridylic acid.

Capping Structures at the 5′-Terminus of Polyadenylated Ribonucleic Acid in Avena Coleoptiles

Evidence is presented for the occurrence of 5'-terminal capping structures in the polyadenylated RNA of oat (Avena sativa) coleoptiles. These structures are composed of an inverted terminal nucleoside containing the modified base 7-methylguanine which is joined 5' to 5' with a second (penultimate) nucleoside by means of three phosphate groups in two pyrophosphate linkages. The penultimate nucleoside is joined to the remainder of the RNA molecule by a conventional 3',5' phosphodiester bond. A significant difference between the cap structures of oat coleoptile RNA and those of previously described higher eucaryotic cellular mRNAs is the lack of ribose methylations in the penultimate nucleosides of the plant RNA.

Wheat embryo ribonucleates. XII. Formal characterization of terminal and penultimate nucleoside residues at the 5'-ends of "capped" RNA from imbibing wheat embryos.

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains[methyl-3H]methionine. (2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of "capped" oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2. (3) By way of contrast with the "capped" RNA of animal cells, 5'-terminal "cap" structures (m7Gppp- and m32,2,7 Gppp-) in the "capped" RNA from the higher plant organism are not bonded to pneultimate O2'-methylnucleoside constituents. (4) In an allied study, it has been found that recovery of poly (A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly (A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris (hydroxymethyl) aminomethane buffered (pH 9.0) phenolic emulsions.

Terminal structure involving a single-stranded stretch in the double-stranded RNA from Penicillium chrysogenum virus

The terminal structure of double-stranded RNA extracted from Penicillium chrysogenum was studied by 3H-labeling with borohydride reduction for the cis-diol in the terminal nucleoside and by 32P-labeling with polynucleotide kinase for the 5′-terminal. Three RNA segments of different molecular weights were equally labeled. The terminal structure was the same for these three RNA segments. The nucleotide sequences near the 3′-terminal were —G-U for one strand and —Y-A for the other. The [ 32P]phosphate was incorporated into only one strand. The labeled 5′-terminus has the sequence A-G-Y—. The 5′-terminus of the other strand seemed to be blocked by something like the confronting nucleotide m 7G 5′ppp 5′N(m)- found in the mRNA strand of other double-stranded RNA viruses, since a 3H-labeled, unidentified nucleotide was observed besides the 3′-terminal nucleoside derivatives. As the 32P-labeled 5′-terminal A-G-Y- is not complementary to either of the two 3′-terminal sequences, the secondary structure was examined by treatment with Aspergillus nuclease S 1, which selectively splits single-stranded parts in polynucleotides. The 32P-labeled 5′-nucleotide was released, whereas the 3H-labeled nucleosides were not released. The nucleotides near the 5′-terminus of the unblocked strand carrying the sequence A-G-Y- must be in a single-stranded stretch, while both the 3′-termini are paired with a complementary strand.

The mechanism by which heparin stimulates transcription in isolated rat liver nuclei. Polyribonucleotide elongation rates and the number of transcribing RNA polymerase molecules present.

The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei; it has been suggested that this process involves either the inactivation of R Nase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 °C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to α‐amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH4)2SO4 and Mn2+ ions. The size of the RNA product was analysed on denaturing formamide sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and α‐amanitin which together inhibit 97% of all RNA synthesis. These results show that inactivation of R Nase cannot be an explanation of the stimulatory effect in this system.Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [3H]UTP during 1‐min incubations. The [3H]UMP and [3H]uridine, derived from internal nucleotides and 3′‐terminal nucleosides respectively, were separated by thin‐layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of R Nase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25–35×103 transcribing RNA polymerase molecules per diploid genome comprising 18–20×103 form A and 5–15×103 form B. Form C was not detected. Addition of heparin has no effect on these values. Polyribonucleotide elongation rates in nuclei are approximately 0.15 and 0.4 nucleotide per second for form A and B respectively. In the presence of heparin the latter rate is increased by 2–4‐fold but that of the form A enzyme remains essentially unchanged. Similar results were obtained using [3H]GTP in place of [3H]UTP indicating that polyribonucleotide termination was random. Our findings show that heparin exerts its stimulatory effect by increasing the form‐B‐mediated polyribonucleotide elongation rate. It does not activate previously inactive RNA polymerase molecules.