RNA Phosphate Research Articles

Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes.

The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates. Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex.

Cyclization of RNA 3'-terminal phosphate by cyclase from HeLa cells proceeds via formation of N(3')pp(5')A activated intermediate.

RNA 3'-terminal phosphate cyclase has been partially purified from HeLa cells. In the presence of ATP and Mg2+, cyclase preparations catalyze conversion of RNA 3'-terminal phosphate to the 2',3'-cyclic phosphodiester. The mechanism of 3'-phosphate cyclization was studied with oligoribonucleotides containing terminal 2'-deoxy- or 2'-O-methylribose. Incubation of these substrates with cyclase and ATP results in formation of the corresponding activated 3'-terminal structures, dN(3')pp(5')A and Nm(3')pp(5')A. It is proposed that an intermediate step in cyclization is transfer of the adenylyl group from ATP to the 3' phosphate of RNA. Rapid attack of the adjacent 2'-OH normally follows, resulting in elimination of AMP and formation of the cyclic phosphodiester. Cyclase preparations can be covalently labeled with [alpha-32P]ATP, suggesting that an earlier step in the cyclization reaction involves formation of an adenylylated enzyme intermediate.

Protein-RNA interactions in belladonna mottle virus investigated by laser Raman spectroscopy.

Raman difference spectroscopy of the belladonna mottle virus (BDMV) and its separated RNA and protein components indicates that molecular interaction occurs between the single-stranded RNA genome and capsid subunits. The molecular interactions that stabilize the virion at pH 5.0 are altered or eliminated at pH 8.0, even though release of the RNA from the capsid is prevented by the addition of divalent metal cations (Ca2+). From the perturbations that occur to Raman lines of cytosine and adenine rings of the encapsidated RNA molecule between pH values of 5.0 and 8.0, it is concluded that cytosines are protonated in significant numbers at the conditions which maintain the native virus structure and that the stacking of adenines is altered by changes in pH. The degree of protonation of RNA bases can be reduced by elevation of the pH to 8.0 for encapsidated RNA or by release of the RNA from the capsid at pH 5.0. Although the protein groups that interact with the viral RNA cannot be identified unambiguously from the Raman spectra, it is apparent that the molecular environments of aromatic amino acid side chains are altered with the same changes in pH (from 5.0 to 8.0) that perturb the cytosine and adenine ring structures. No significant change in secondary structures of the capsid subunit can be detected with changes in pH or with RNA release. On the other hand, the characteristic Raman lines of the phosphate groups of packaged RNA differ from those of naked RNA at all pH values examined, most likely as a result of specific electrostatic binding of divalent cations to RNA phosphates within the virus shell.

Phosphorus-31 nuclear magnetic resonance study of internal motion in ribonucleic acid of southern bean mosaic virus.

Phosphorus-31 nuclear magnetic resonance spectroscopy was used to study southern bean mosaic virus (SBMV) in its native and ethylenediaminetetraacetic acid treated forms at various pH values in the range 4.5-9.0. The degree of swelling in the latter form is a critical function of pH. Spin-lattice relaxation time (T1), line-width, and nuclear Overhauser effect (NOE) measurements were made at 40.51, 80.98, and 190.3 MHz. The results indicate that the RNA phosphate groups in native SBMV exhibit low-amplitude internal motions on a nanosecond time scale while the phosphate groups of swollen virus exhibit large-amplitude, more rapid, internal motions. A spread of T1 and NOE values is measured at intermediate stages in the swelling process. A simple explanation, consistent with the data, is that the RNA core of the virus undergoes a two-state phase transition from a relatively rigid, compact solid form in the native SBMV to a mobile, solvated state in the fully swollen particle.

Mechanism of RNA-protein interactions in tobacco mosaic virus: analysis of the pH stability of virus protein complexes with synthetic polynucleotides.

TMV-like RNP complexes were reconstituted from TMV protein and synthetic polynucleotides. Analysis of the pH stability of RNP with polynucleotides containing U, G, or their analogues reveals a correlation between the stability of their structure and the pK values of the bases, and indicates that the -NH-CO-groups of U and G are involved in hydrogen bonding with protein. It is suggested that TMV protein has two U- and one G-specific binding sites which, according to the phase position of the protein subunits relative to the origin of TMV assembly (D. Zimmern (1977), Cell 11, 463) are likely to be organized as UGU. The binding of the A and C residues of RNA with TMV protein is nonspecific. TMV protein groups with pK 6.3, 7.5 and 9.7 were found to be essential in the protein-protein interactions in RNP. A group of the protein with pK 8.2 is also involved in RNP stabilization. Both protein-protein interactions and interactions of protein with RNA phosphate groups were shown to be mediated by a conformational change in the protein induced by base binding. The effect of bases on both types of interactions changes in the order G approximately equal to much greater than A, and incorporation of C in RNP proceeds in a compulsory way at the expense of interaction of the neighbouring nucleotide residues in polynucleotides with protein. The data obtained are used to discuss the principles of the cooperativity of the interactions between TMV components and the mechanism of initiation and elongation in TMV self-assembly.

The effect of clupein on anisotropy and basophilia of polytene chromosomes.

When polytene chromosomes are subjected to a clupein treatment, their properties of basophilia and anisotropy are affected. The basophilia is deeply reduced, except in the nucleolar zones, puffs and sites of RNA accumulation. On the other hand, the chromosome birefringence increases. The phenomenon of anomalous dispersion of birefringence usually observed on polytene chromosomes stained with toluidine blue solutions turns into a normal negative dispersion of birefringence, when staining is preceded by clupein treatment. It is concluded that the clupein molecules attach orderly and preferentially to sequential DNA phosphates unbound to chromosome proteins, accentuating DNA anisotropic characteristics. The clupein molecules appear not attaching to RNA phosphates.

Potato spindle tuber viroid: circular dichroism spectrum and physical chemical studies of its interaction with ethidium bromide.

The presence and extent of base-paired secondary structure in the protein-free potato spindle tuber viroid RNA has been investigated by spectroscopic techniques. Dye-binding studies with the intercalating dye, ethidium bromide, indicate the presence of base-paired regions in the molecule. Variation of the induced chirality in the dye with the dye to RNA phosphate ratio indicate the presence of two base-paired regions in the molecule.

The cation binding associated with structural transitions in bromegrass mosaic virus

Hydrogen ion titration curves are described for bromegrass mosaic virus under various conditions of temperature, ionic strength, and Ca 2 +, Mg 2+, or polyamine concentration, with particular reference to the hysteresis region near neutral pH. The curves are correlated with the sedimentation properties of the virus. There are two relatively nonspecific cation-binding sites per protein subunit, present in the virion but not in the protein or RNA alone, which control the virion's swelling. The swelling is reversible in the presence of enough cations; without them, swollen BMV can contract to an incorrect structure with RNA partially exposed. We interpret these results qualitatively and structurally in terms of binding sites formed in a region of high electronegativity contributed by RNA phosphates and protein carboxylates.

ChemInform Abstract: ALL OXYGENS IN NUCLEIC ACIDS REACT WITH CARCINOGENIC ETHYLATING AGENTS

Over 80% of ethylnitrosourea and ethylnitrosoguanidine modification of nucleic acids is on oxygens. The reactivity of oxygens (other than ribose and phosphate) in single-stranded RNA is: O2 of C greater than O2 of U greater than O6 of G greater than O4 of U. In double-stranded DNA the order is: O2 of T equals O6 of G greater than O4 of T greater than O2 of C. Oxygen reactivity of single-stranded DNA resembles RNA. The glycosidic bond of O2-alkylpyrimidines is labilised.

All oxygens in nucleic acids react with carcinogenic ethylating agents.

Over 80% of ethylnitrosourea and ethylnitrosoguanidine modification of nucleic acids is on oxygens. The reactivity of oxygens (other than ribose and phosphate) in single-stranded RNA is: O2 of C greater than O2 of U greater than O6 of G greater than O4 of U. In double-stranded DNA the order is: O2 of T equals O6 of G greater than O4 of T greater than O2 of C. Oxygen reactivity of single-stranded DNA resembles RNA. The glycosidic bond of O2-alkylpyrimidines is labilised.

Incorporation of phosphorus into phased cells ofCandida utilis using32P and33P

Changes in phosphorus metabolism were studied by examining the incorporation of32P and33P into cells ofCandida utilis growing in phased culture during a 6 h cell cycle and a post-cycle period of 6 h. Three different chemically defined media were used; these were phosphorus, nitrogen and carbon limited. The patterns of incorporation of phosphorus into RNA, DNA, lipid and cold water extractable phosphate fractions showed a non-uniform behaviour during both cell cycle and post-cycle periods. The patterns were different in all three types of media. The results showed that a cell can grow and develop at a fixed growth rate in different ways: so that the pattern of behaviour during a cell cycle is not stereotyped for a given doubling time, but largely depends upon the nutrient environment in which the cell exists.

Purification and Properties of Acid Deoxyribonucleases of Human Gastric Mucosa and Cervix Uteri

Abstract The acid deoxyribonucleases in homogenates of human gastric mucosa and cervix uteri were purified extensively by a procedure including DEAE-cellulose and phosphocellulose chromatographies, gel filtration, and isoelectric focusing. The enzymes in the two preparations had similar properties. The active protein was resolved into at least two forms with different isoelectric points, as judged by the formation of acid-soluble fragments. The major component of the activity had an isoelectric point at pH 7.02, and the minor component at pH 6.86. However, other properties of these two forms were similar. The final preparations of the gastric and uterine acid DNases were free of other types of DNase (DNases I, III, and IV), acid and alkaline phosphatases, acid and basic RNases, and nonspecific phosphodiesterase. Both enzymes had a molecular weight of approximately 38,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 5.2 to 5.5 in 33 mm acetate buffer. They did not require divalent cations for activity, and hydrolyzed native, double-stranded DNA about 10 to 20 times faster than heat-denatured DNA. They did not act on RNA or calcium bis(p-nitrophenyl) phosphate, indicating that they did not contain intrinsic phosphodiesterase activity. The products formed from native DNA were 3'-phosphoryl- and 5'-hydroxyl-terminated oligonucleotides. The average chain length of the limit digests with these enzymes was approximately 10 and the predominant species were longer than hexanucleotides.

Occurrence of polyamines in myxoviruses.

SUMMARY Newcastle disease and influenza viruses were purified by centrifuging in sucrose and potassium tartrate gradients and analysed for polyamines by thin-layer chromatography of dansyl derivatives. It has been shown that both viruses contained spermine, spermidine and putrescine in concentrations sufficient to neutralize 30 to 40% of virus RNA phosphates.

Effect of temperature on the cellular integrity of Bacillus psychrophilus.

The release of intracellular materials from the psychrophile Bacillus psychrophilus at temperatures from 5 to 50C was studied as a means of indicating possible membrane damage. RNA, protein, carbohydrate, and inorganic phosphate, but not DNA was found to leak from the cells. The rate of leakage increased as the temperature was raised. It is shown that the amount of leakage at 40C in the length of time required for 90% death could be sustained at 25C with little or no death. Therefore, death of this psychrophile at supramaximal temperatures cannot be attributed to loss of membrane integrity with resultant loss of intracellular material. Electron micrographs of the cells demonstrated a trilaminar cell wall which becomes severely damaged during heating. The damage is irreversible at 40C and may contribute to heat-induced death.

Histochemical aspects of the differentiation of adventitious cartilage on the membrane bones of the embryo chick.

The histochemistry of the adventitious cartilage of the chick has been studied and compared with both primary cartilage and the bone on which the adventitious cartilage develops. The distribution of DNA, RNA, collagen, acid mucopolysaccharide, mucoprotein, glycogen, lipid, alkaline phosphatase and inorganic phosphate has been studied. Adventitious cartilage was found to have the histochemistry of primary hypertrophic cartilage and to calcify. The appearance of lipid and alkaline phosphatase activity coincided with the onset of calcification.

Studies on extracellular ribonucleases of Ustilago sphaerogena. Characterization of substrate specificity with special reference to purine-specific ribonucleases.

1. Ribonuclease U(1) splits only the phosphodiester bonds of guanosine 3'-phosphates in RNA. It may be regarded as a guanyloribonuclease [ribonucleate (guanine nucleotide)-2'-transferase (cyclizing), EC 2.7.7.26] similar to ribonuclease T(1) (Egami, Takahashi & Uchida, 1964). It seems to be identical with the extracellular ribonuclease described by Glitz & Dekker (1963, 1964a,b). 2. Ribonucleases U(2) and U(3) are novel enzymes with a strict specificity. They split the internucleotide bonds between purine 3'-nucleotides and 5'-hydroxy groups of adjacent nucleotides in RNA with the intermediary formation of purine nucleoside 2',3'-(cyclic)-phosphates, which are slowly hydrolysed to purine 3'-nucleotides. So they may be classified as ;puryloribonucleases [ribonucleate (purine nucleotide)-2'-transferase (cyclizing)]'. Double-stranded RNA is scarcely split by ribonucleases U(2) and U(3). 3. Ribonuclease U(4) has no absolute base specificity, and produces the mononucleotides 3'-adenylate, 3'-guanylate, 3'-cytidylate and 3'-uridylate from RNA.

Structural studies on ribosomes: I. The binding of proflavine to Escherichia coli ribosomes

The binding of proflavine to 30-S, 50-S and 70-S ribosomes has been studied quantitatively utilising spectral changes in the dye to measure the degree of binding. All three particles produced hypochromism in the ultraviolet difference spectrum of the dye and a displacement in the visible spectrum to longer wavelengths with the formation of characteristic isobestic points. An analysis of the spectral changes revealed two distinct binding processes. A strong binding characterised by a free energy of association of 10 kcal/mole appeared to involve Van der Waals interactions between the dye and the bases on the RNA; a weak interaction was attributed to electrostatic binding of the dye to the RNA phosphates. The number of strong binding sites was 96, 70 and 108 for the 30-S, 50-S and 70-S ribosomes, respectively. Despite an increase of about 50 in the number of binding sites when 70-S ribosomes dissociate into their subunits the binding of proflavine to 30-S and 50-S ribosomes did not appear to affect the reassociation of these particles to form 70-S ribosomes. The number of strong binding sites on the 50-S ribosome increased to about 500 when the particle was unfolded in EDTA. This suggested that binding sites, which are exposed in the unfolded particle, are not available for binding dye in the intact ribosome because of the rigid tertiary folding in the native particle.

Stimulation of polyamine synthesis in relation to nucleic acids in regenerating rat liver

The effect of partial hepatectomy on the liver polyamines and nucleic acids was studied in the rat. Partial hepatectomy causes an early stimulation of spermidine synthesis, as indicated by the marked increase in the incorporation of [ 14C]methionine into this polyamine, as early as 4–8 h after operation. At 16 h the specific activity of spermidine was about 10-fold that of the sham-operated controls. The total spermidine content of the liver was already significantly elevated at 16 h, and at 64 h it was 3.6-fold that of the controls. The specific activity of spermine, on the other hand, did not increase until 16–20 h and the total amount of spermine per liver not until 64 h post-operatively. The total amount of liver ribonucleic acid was significantly increased at 32 h and that of deoxyribonucleic acid at 64 h. The ratio polyamine nitrogen to RNA phosphate remained quite constant during liver regeneration. The present observations are discussed on the basis of the hypothesis that tissue polyamines may be essential as physiological stabilizers of nucleic acids, especially RNA.

Magnesium binding by Escherichia coli ribosomes

Magnesium binding by ribosomes and ribosomal RNA from Escherichia coli has been studied as a function of the free magnesium concentration. At 10−2M-Mg2+ the 70 s and 100 s components bind at least three times more magnesium than they do at 10−4M-Mg2+ when they are completely dissociated into their 30 s and 50 s subunits. Between 10−4M- and 10−2M-magnesium, a pair of 30 s and 50 s particles take up about 1600 magnesium ions, thus approaching a ratio of bound Mg2+ to P of 0·5. Increasing concentrations of potassium decrease the extent of magnesium binding by competition for anionic sites. Magnesium is nevertheless bound preferentially, even at potassium to magnesium ratios greater than 10 to 1. Parallel studies have been carried out on the magnesium binding of ribosomal RNA. These studies support the suggestion of Ts'o and his co-workers that virtually all the magnesium bound by the ribosomes is bound by the RNA. Binding to a greater extent than one magnesium atom for every two RNA phosphates has been observed, but this effect appears to be correlated with the precipitation of the RNA at high ionic strengths. Almost all of the phosphate groups of the RNA must be available for magnesium binding in the completed ribosome, for removal of the protein moiety makes available at most 12% more binding sites.

Hart-Valentine培地中におけるそらい菌の菌長, RNA, DNA及び無機燐の消長

Hart-Valentine培地中におけるそらい菌の菌長, RNA, DNA及び無機燐の消長