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PubMed1980

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei.

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.

Adenosine Triphosphatases↗
PubMed1976

Conformational properties of dinucleoside monophosphates in solution: dipurines and dipyrimidines.

In order to obtain information about the conformational features in a polyribonucleotide at the nearest neighbor level, detailed nuclear magnetic resonance studies of the dinucleoside monophosphates ApA, ApG, GpA, UpU, CpC, UpC, and CpU were undertaken. Proton spectra were recorded at 100, 220, 270, or 300 MHz for D2O solutions, 0.01-0.03 M, pD 7.4 at 20+/-2 degrees C. Spectra of ApA, ApG, UpU, and UpC were also recorded in the temperature range of 70-90 degrees C. Unambiguous signal assignments of all proton resonances were made with the aid of selectively deuterated dimers. Complete, accurate sets of nuclear magnetic resonance (NMR) parameters were derived for each nucleotidyl unit by simulation-iteration methods. A complete set of chemical shift and coupling constant data was also obtained for all the constituent monomeric units at a concentration and ionization state comparable to that of the dimers. Conformational properties were evaluated quantitatively for most of the bonds in the dinucleoside monophosphates using procedures developed in earlier studies. All of the dimers have a flexible conformational framework in aqueous solution. While flexibility is allowed and alternate conformations are accessible, these molecules nevertheless attempt to achieve conformational identity by showing preferences--sometimes overwhelming preferences--for certain orientations. Thus the ribose rings exist as equilibrium mixtures of C2'-endo in equilibrium C3'-endo conformers with a bias for the C3'-endo pucker in most cases. The C4'-C5' bonds of both nucleotidyl units show significant preference (70-85%) for a gg conformation. Similarly, the dominant conformer (80-90%) about C5'-O5' is g'g'. Even though an unambiguous determination of the orientation about C3'-O3' cannot be made, there is suggestive evidence that the orientation of the 3' phosphate group is coupled to the ribose conformational equilibrium and it is likely that a 3Eg- in equilibrium 2Eg+ equilibrium exists with a bias for the 3Eg- coupled conformation in which the H3'-C3'-O3'-P dihedral angle is about 34-38 degrees. The individual nucleotidyl units in the dimers differ in several key ways from corresponding monomer conformations. Specifically, the ribose equilibrium C2'-endo in equilibrium C3'-endo shifts in favor of C3'-endo upon dimerization, the only exception being UpU. The C4'-C5' and C5'-O5' bonding network in the dimer forms a stable conformational unit and no correlation exists in the dimers between the conformational preference of this fragment and ribose conformer population. The temperature data for the dimers and dimerization data clearly indicate that the transition C2'-endo leads to C3'-endo is directly related to XCN changes brought about by dimerization and stacking...

Magnetic Resonance Spectroscopy↗
PubMed1976

Alkylation of ribose in RNA reacted with ethylnitrosourea at neutrality.

Ribose oxygens in TMV-RNA are ethylated by the carcinogen ethylnitrosourea in neutral aqueous solution (pH 6.1-7.3). 2'-O-ethyluridine, and 2'-O-ethylcytidine have been identified as reaction products. The four 2'-O-ethyl nucleosides are found in approximately equal amounts and the total extent of ribose alkylation is about 15% of the total ethylation. This finding, in conjunction with earlier results showing that all ring and phosphate oxygens can be ethylated, signifies that every oxygen in RNA or polyribonucleotides can react with ethylnitrosourea. The possible biological significance of ribose alkylation, resulting from chemical rather than enzymatic reaction, is discussed. The preparation of the new derivative 2'(3')-Oethylguanosine is described.

Alkylation↗
PubMed1975

Association of the polyadenylate segment of messenger RNA with other polynucleotide sequences in mouse sarcoma 180 polyribosomes.

Limited digestion of polysomal RNA with pancreatic ribonuclease releases a structure consisting of poly(A) associated with other polyribonucleotide sequences. This complex can be purified by oligo(dT)-cellulose chromatography. Heating for formamide treatment causes the dissociation of fragments free of poly(A) from the poly(A)-containing components. The two types of fragments tend to reassociate under annealing conditions, and this association is prevented by poly(U). Control experiments indicate that this structure is not an artifact generated during the manipulations. The same structure can be obtained by limited RNase digestion of polyribosomes, followed by deproteinization. The results suggest that the mRNA in polyribosomes may have a defined configuration caused by the interaction of the poly(A) sequence with another segment of the RNA.

Adenine↗
PubMed1975

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.

Animals↗
PubMed2004

Selective derivatization and sequestration of ribose from a prebiotic mix.

Observations regarding the catalytic potential of RNA and the role of RNA in biology have formed the basis for the "RNA world" hypothesis, which suggests that a genetic system based on self-replicating polyribonucleotides preceded modern biology. However, attempts to devise a realistic prebiotic synthesis of nucleic acids from simple starting materials have been plagued by problems of poor chemical selectivity, lack of stereo- and regiospecificity, and similar rates of formation and degradation of some of the key intermediates. For example, ribose would have been only a small component of a highly complex mix of sugars resulting from the condensation of formaldehyde in a prebiotic world. In addition, ribose is more reactive and degrades more rapidly compared with most other monosaccharides. This study demonstrates an approach for the preferential sequestration of ribose relative to other sugars that takes advantage of its greater reactivity. Cyanamide reacts especially rapidly with ribose to form a stable bicyclic adduct. This product crystallizes spontaneously in aqueous solution, whereas the corresponding products derived from threose, galactose, glucose, mannose, and each of the other pentoses do not. Furthermore, when employing a racemic mixture of d- and l-ribose, enantiomerically twinned crystals are formed that contain discrete homochiral domains.

Cyanamide↗
PubMed2002

The hydrogen bonding and hydration of 2'-OH in adenosine and adenosine 3'-ethyl phosphate.

The 2'-OH group has major structural implications in the recognition, processing, and catalytic properties of RNA. We report here intra- and intermolecular H-bonding of 2'-OH in adenosine 3'-ethyl phosphate (1), 3'-deoxyadenosine (2), and adenosine (3) by both temperature- and concentration-dependent NMR studies, as well as by detailed endo ((3)J(H,H)) and exocyclic ((3)J(H,OH)) coupling constant analyses. We have also examined the nature of hydration and exchange processes of 2'-OH with water by a combination of NOESY and ROESY experiments in DMSO-d(6) containing 2 mol % HOD. The NMR-constrained molecular modeling (by molecular mechanics as well as by ab initio methods both in the gas and solution phase) has been used to characterize the energy minima among the four alternative dihedrals possible from the solution of the Karplus equation for (3)J(H2',OH) and (3)J(H3',OH) to delineate the preferred orientation of 2'-O-H proton in 1 and 2 as well as for 2'/3'-O-H protons in 3. The NMR line shape analysis of 2'-OH gave the DeltaG(H-bond)(298K) of 7.5 kJ mol(-1) for 1 and 8.4 kJ mol(-1) for 3; similar analyses of the methylene protons of 3'-ethyl phosphate moiety in 1 also gave comparable DeltaG(H-bond)(298K) of 7.3 kJ mol(-1). The donor nature of the 2'-OH in the intramolecular H-bonding in 3 is evident from its relatively reduced flexibility [-TDeltaS++](2'-OH) = -17.9(+/-0.5) kJ mol(-1)] because of the loss of conformational freedom owing to the intramolecular 2'O-H...O3' H-bonding, compared to the acceptor 3'-OH in 3 [-TDeltaS++](3'-OH) = -19.8 (+/- 0.6) kJ mol(-1)] at 298 K. The presence of intramolecular 2'-OH...O3' H-bonding in 3 is also corroborated by the existence of weak long-range (4)J(H2',OH3') in 3 (i.e., W conformation of H2'-C2'-C3'-O3'-H) as well as by (3)J(H,OH) dependent orientation of the 2'- and 3'-OH groups. The ROESY spectra for 1 and 3 at 308 K, in DMSO-d(6), show a clear positive ROE contact of both 2'- and 3'-OH with water. The presence of a hydrophilic 3'-phosphate group in 1 causes a much higher water activity in the vicinity of its 2'-OH, which in turn causes the 2'-OH to exchange faster, culminating in a shorter exchange lifetime (tau) for 2'-OH proton with HOD in 1 (tau2'-OH: 489 ms) compared to that in 3 (tau2'-OH: 6897 ms). The activation energy (E(a)) of the exchange with the bound-water for 2'- and 3'-OH in 3 (48.3 and 45.0 kJ mol(-1), respectively) is higher compared to that of 2'-OH in 1 (31.9 kJ mol(-1)), thereby showing that the kinetic availability of hydrated 2'-OH in 1 for any inter- and intramolecular interactions, in general, is owing to the vicinal 3'-phosphate residue. It also suggests that 2'-OH in native RNA can mediate other inter- or intramolecular interactions only in competition with the bound-water, depending upon the specific chemical nature and spatial orientation of other functions with potential for hydrogen bonding in the neighborhood. This availability of the bound water around 2'-OH in RNA would, however, be dictated by whether the vicinal phosphate is exposed to the bulk water or not. This implies that relatively poor hydration around a specific 2'-OH across a polyribonucleotide chain, owing to some hydrophobic microenvironmental pocket around that hydroxyl, may make it more accessible to interact with other donor or acceptor functions for H-bonding interactions, which might then cause the RNA to fold in a specific manner generating a new motif leading to specific recognition and function. Alternatively, a differential hydration of a specific 2'-OH may modulate its nucleophilicity to undergo stereospecific transesterification reaction as encountered in ubiquitous splicing of pre-mRNA to processed RNA or RNA catalysis, in general.

Adenosine↗
PubMed2002

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme.

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg2+ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.

Amino Acid Sequence↗
PubMedDate not supplied

Formation of a 2'-phosphomonoester, 3',5'-phosphodiester linkage by a novel RNA ligase in wheat germ.

Extracts of wheat germ contain an RNA ligase activity that catalyses the conversation of linear polyribonucleotides into covalently closed circles. The newly formed 3',5'-phosphodiester bridge is resistant to alkali and a number of ribonucleases. This unusual stability is due to the presence of a phosphoryl group esterified to the 2'-position of the 3'-nucleotide joined. This is the first demonstration of an RNA ligase in higher plants and of a natural 2'-phosphomonoester, 3',5'-phosphodiester linkage.

Nucleic Acid Precursors↗
PubMed1994

An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis.

The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

3T3 Cells↗
PubMed1976

Effects of dibutyrylcyclic AMP on the in vitro primary response of mouse spleen cells to sheep erythrocytes.

Exogenously added N6, 02'-dibutyryladenosine-3'-5'-cyclic phsophate (DBcAMP) exerted a biphasic effect on the in vitro primary response of mouse spleen cells to sheep erythrocytes (SRBC). High concentrations of DBcAMP were markedly suppressive, whereas lower concentrations brought about significant increases in the peak numbers of antibody-forming cells. The kinetics of the response were not altered by DBcAMP. A direct effect of DBcAMP on bone-marrow derived lymphocytes was suggested because this nucleotide increased the in vitro response of "nude" mouse spleen cells to SRBC. The magnitude of the response, however, was considerably less than that mounted by spleen cells of normal mice. These results show that DBcAMP can have either enhancing or suppressive effects on antibody formation and these effects are dependent upon nucleotide concentration, antigen-dosage and time of assay. The effects of this cyclic nucleotide differed in two major respects from those of the synthetic polyribonucleotide adjuvant, polyadenylic acid-polyuridylic acid (poly A:U) complexes. Thus poly (A:U) decreased the time of peak response in vitro but did not increase the magnitude of the response.

Animals↗
PubMed1967

The incorporation of cytidine 5'-monophosphate and other ribonucleotides by an enzyme preparation from rat-liver microsomes.

1. An enzyme preparation from rat-liver microsomes incorporated all four ribonucleotides from the corresponding triphosphates into ribosomal RNA. The reaction was Mn(2+)-dependent, but UMP incorporation also occurred in the presence of Mg(2+). 2. The incorporation of any one ribonucleotide was inhibited by the presence of the other three ribonucleoside triphosphates and by denatured DNA. 3. The product of the reaction consisted of short chains of homopolymer attached to the primer ribosomal RNA. 4. ;Soluble' RNA, synthetic polyribonucleotides, and oligoribonucleotides were also effective primers for CMP incorporation. 5. When phosphodiesterase-treated ;soluble' RNA was the primer, CMP was incorporated into positions usually occupied by the normal terminal trinucleotide sequence of intact ;soluble' RNA, but the enzyme did not synthesize a specific terminal sequence consisting of a defined number of CMP residues.

Adenosine Triphosphate↗
PubMed1967

The conformation of ribonucleic acids in Escherichia coli ribosomes. Inferences from the mode of action of ribonuclease II.

1. Ribonuclease II of Escherichia coli degrades pulse-labelled RNA associated with ribosomes and polyuridylic acid on ribosomes and in solution to mononucleotides. 2. Ribosomal and pulse-labelled RNA in solution and ribosomal RNA in chloramphenicol particles (protein-deficient ribosomes) are degraded to oligonucleotides. 3. Ribosomal RNA in mature ribosomes is not attacked by the enzyme. 4. From the mode of action of ribonuclease II, which is specific for single-stranded polyribonucleotides and does not attack helical forms, it is inferred that pulse-labelled RNA associated with ribosomes of E. coli exists as a single-stranded structure and that ribosomal RNA in chloramphenicol particles has a pronounced helical character. 5. The different behaviour of ribonuclease II towards newly synthesized RNA, ribosomal RNA and chloramphenicol-particle RNA in E. coli ribosomes is discussed.

Carbon Isotopes↗
PubMed1969

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis.

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.

Centrifugation, Density Gradient↗
PubMed1969

The influence of the size and nature of basic activators on Clostridium perfringens polynucleotide phosphorylase-catalysed polyadenylic acid synthesis.

1. Basic oligo- and poly-(amino acids) stimulate polyadenylic acid synthesis by purified Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8). 2. The effectiveness of the activators increases with chain length up to approx. 20-30 residues. 3. Polymers of the l and dl series are equally effective on a weight-for-weight basis. 4. l-Lysine, d-lysine, diethylamine and triethylamine, as hydrochlorides or hydrobromides, all stimulate the reaction markedly if their concentration is high enough. Their effect is similar to that of sodium chloride. 5. The size of the product depends primarily on the Mg(2+) concentration and basic polymers have a relatively limited effect on it. 6. Polyadenylic acid itself undergoes an Mg(2+)-catalysed non-enzymic hydrolysis.

Acrylates↗
PubMed1970

Conformation of nucleic acids and the analysis of the hypochromic effect.

The spectra of two double-helical RNA species isolated from virus-like particles found in the mycelium of Penicillium chrysogenum were measured at about 25 degrees C, and at 95 degrees C after denaturation to a single-stranded form, and compared with the spectrum of the equivalent mixture of nucleotides. For both species the difference spectrum (epsilon(95 degrees C)-epsilon(25 degrees C)) when increased by 33% was very similar to the difference spectrum (epsilon(nucleotides)-epsilon(25 degrees C)). In contrast it was found for rat liver DNA that there was no simple relation between (epsilon(95 degrees C)-epsilon(25 degrees C)) and (epsilon(nucleotides)-epsilon(25 degrees C)). These observations were accounted for on the basis of the difference spectra for rA.rU, rG.rC, dA.dT and dG.dC base-pairs derived from model polyribonucleotides and polydeoxyribonucleotides. The difference spectra (epsilon(95 degrees C)-epsilon(25 degrees C)) found for rRNA and those calculated from a combination of the difference spectra for rA.rU and rG.rC base-pairs were in good agreement.

Animals↗
PubMed1972

Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum.

1. The RNA-dependent RNA polymerase from Halobacterium cutirubrum was purified to electrophoretic homogeneity. 2. It requires a single-stranded molecule of RNA or polyribonucleotide as template. 3. Nearest-neighbour analyses of the products formed on random poly(A,U) or alternating poly(A-U) templates and base analysis of the product of synthesis directed by wheat-germ RNA prove that the template is copied accurately. 4. The enzyme initiates new chains with purine ribonucleoside triphosphates. 5. Sucrose-density-gradient analysis of the product indicates that it has a size distribution similar to that of the template. 6. Preliminary amino acid analysis of the RNA-dependent polymerase shows that it contains much less serine than either of the subunits of H. cutirubrum DNA-dependent RNA polymerase. 7. The RNA-dependent enzyme is unable to substitute for either subunit of the DNA-dependent polymerase, and both the latter are devoid of RNA-dependent activity.

Amino Acids↗
PubMed1973

Association of nascent polypeptide with 30S ribosomal subunits.

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg(2+) concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).

Amino Acids↗