Nucleic Acid Conformation Research Articles

Nucleic acid conformation: crystal structure of a naturally occurring dinucleoside phosphate (UpA).

THEORIES of the molecular structure of nucleic acids have so far been based on evidence from the crystal structures of monomeric units such as nucleosides and mononucleotides, the interpretation of diffraction patterns of oriented nucleic acid fibres and molecular model building1–6. Such approaches can help to suggest structures of periodic molecules such as helices, but they are insufficient for predicting and understanding nonrepetitive structures such as the loops in transfer RNA (tRNA), presumably associated with many of the functions of tRNA. To understand the geometry of nucleic acids and possible constraints on their conformation, it is therefore essential to know the detailed conformation of the sugar residues and the conformational relationship between the sugar residue, the base and the phosphate group7–9. The simplest molecule which contains this information is a 3´5´-dinucleoside phosphate. We now report the structure of uridine-3´,5´-adenosine phosphate (UpA). This is the first naturally occurring dinucleoside phosphate whose crystal structure has been determined by X-ray diffraction. The only other dinucleoside phosphate with known crystal structure is adenosine-2´,5´-uridine phosphate10, but it does not have the naturally occurring 3´5´ sugar phosphate linkage.

Molecular orbital calculations on the conformation of nucleic acids and their constituents: I. Conformational energies of β-nucleosides with C(3′)- and C(2′)-endo sugars

Molecular orbital calculations, using the all-valence-electrons PCILO method, are carried out for the evaluation of conformational energies of purine and pyrimidine nucleosides as a function of the rotation about the C-N glycosidic linkage. In distinction to previous empirical treatments which were based on the partitioning of the potential energy into different constituents and a separate evaluation of those, the quantum-mechanical computations provide directly the total molecular energy corresponding to the different geometrical arrangements of the atoms. Explicit distinction is also made in the calculations for cases in which an intramolecular hydrogen bond is or is not formed between the sugar and base. Evaluation of the conformation energies is completed by evaluation of the relative probabilities of finding the nucleosides in the anti or syn regions. Results are presented for purine and pyrimidine glycosides with C(3′)-endo and C(2′)-endo sugars and compared with the available experimental data from X-ray studies on crystals.

Interaction of nucleic acids: VIII. Binding of magnesium ions by nucleic acids

The lowering of the activity of magnesium ions in aqueous solution due to the presence of nucleic acids (5 to 20 m m) was measured by a divalent cation specific electrode which shows a large decrease in potential. The electrode response over a wide range of MgCl 2 concentration (10 −1 to 10 −4 m) in water is linear as described by the Nernst equation. The binding studies were carried out in ~ 5 m m-sodium phosphate buffer, at neutral pH, with a variety of nucleic acids. The results are treated as site-binding to the phosphate in a multiple equilibrium using the equation of Scatchard (1949). The binding isotherms in the Scatchard plot (v̄/A 0 versus v̄, where v̄ is the degree of saturation of binding sites and A 0 is the concentration of free Mg 2+) show a limited linear portion from approximately 30 to 70% saturation of binding sites and then the slopes decrease markedly. As a first-order approximation to account for the interference factor (electrostatic, conformational, etc.) in the binding process of magnesium ions by the polymer with only one type of binding site (the phosphate ligand), two additional treatments were added to the Scatchard equation: (1) the binding constant, k, is replaced by ke −φv where φ represents the interference factor; (2) the derivative of the equation was taken to construct a [(δ v ̄ /A 0)/δ v ̄ ] n versus v̄ plot. In these plots, there is a region of zero slope (horizontal region) and a region of negative slope(s). The value from the horizontal region is taken as the apparent intrinsic constant for magnesium ion binding by the nucleic acids in the neutral sodium phosphate buffer at room temperature: transfer RNA (≈1.7 × 10 4) > poly A ≈ poly A· poly U ≈ poly I ≈ poly I · poly C ≈ native DNA ≈ stabilized stored denatured DNA (≈6 × 10 3) > freshly denatured DNA ≈ poly U ≈ poly C ≈ (≈3 × 10 3) ⪢ monomers (≈2 × 10 2). The slope in the derivative plot is related to the quantity −φ, varying from −φ to −2φ. The slopes for all the nucleic acids are similar except for transfer RNA, which has a much larger slope. These results indicate the importance of the conformation of nucleic acid in the binding of magnesium ions as reflected in both the apparent binding constant and the interference factor. Since the binding constant and the interference factor of transfer RNA are larger than those of other nucleic acids, these observations indicate that transfer RNA has a more folded conformation.

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.

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids.

AbstractThe interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly‐nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de‐naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic‐costacking mechanism. Results from studies on optical rotation and helix‐coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg++ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).

Stereochemistry of nucleic acids and polynucleotides. VI. Minimum energy conformations of dimethyl phosphate

AbstractAs part of a study on the conformation of polynucleotides and nucleic acids the preferred conformations of the model conpound dimethyl phosphate are worked out using potential energy functions. In calculating the total potential energy associated with the conformation, nonbonded, torsional, and electrostatic terms have been considered. The variation of the total conformational energy is represented as a function of two torsion angles ϕ and ψ which are the rotations about the two phosphoester bonds. The most stable conformations are found to be the gauche–gauche conformations about these bonds. The conformations observed for phosphodiesters in the solid state and in the proposed structures of polynucleotides and nucleic acids cluster around the minimum. Also, regions of minimum energy correspond well with the typical allowed regions of a representative dinucleotide.

Conformation of Viral Nucleic Acids in Situ

Publisher Summary The problems that are discussed in this chapter are confined to viruses, and to be more exact, to the question of similarities and differences between the structure and properties of usual forms of nucleic acids in solution and that of nucleic acids inside viral particles. Such differences should exist, as the viral particle is a rather peculiar and highly specialized object designed for storage and transport of the genetic substance of viruses. The properties of nucleic acids in situ ought to be affected by the environment, which they have in viruses, by the special functions that they have to perform, by their close partnership with protein, and by a number of other intraviral factors of which very little is known. The bulk of the information dealt with in this chapter is obtained by the means of X-ray diffraction and electron microscopy, various combinations of optical methods, and chemical and physical modifications combined with some way of recording corresponding results. There are a number of theoretical and practical difficulties to be overcome, which are discussed in detail in this chapter. Determination of true absorption, values of objects that scatter light in the region where they absorb, is always a matter of difficulty when conformation of intraviral nucleic acid is studied by means of absorption, anisotropic absorption, or optical rotatory dispersion (ORD). The use of the extrapolation method is often objected to form a theoretical point of view. The more specific problem of obscurity of UV dichroism data and some others are considered in this chapter.

The Role of Conformation in Chemical Mutagenesis

Publisher Summary This chapter discusses in vitro chemical point modifications of nucleic acids and the mutations detectable after such treatments. It describes the chemistry of mutagenic reactions and illustrates the working hypothesis that mutagenesis requires a tautomeric shift in the N-1, C-6 region of the purines or the N-3, C-4 region of the pyrimidines. The chapter explains that the efficiency of a mutagen is dependent on the extent of tautomeric shift it produces and on the ratio of the frequencies of the mutagenic to the inactivating events occurring simultaneously. The chapter summarizes the literature dealing with in vitro mutagenesis, with particular reference to nucleic acid conformation and milieu. It also indicates the relative reactivity of the bases, based on the chemical data. The chapter explains that mutagenesis is usually observed at an early stage of the reaction, before the base changes reach the level of analytical detectability for most reactions.

Possible conformations of 5-S ribosomal RNA.

THE determination of the nucleotide sequence of two 5S ribosomal RNAs by Brownlee, Sanger and Barrell represents a major advance in our knowledge of the structure of ribonucleic acids1. This RNA contains only the four normal bases A, U, C and G, and so it is an ideal system on which to test some of the physico-chemical techniques known to be sensitive to the conformation of nucleic acids in solution2–4. There is a wide range of plausible secondary structures which can be devised for 5S ribosomal RNA. The model conformation suggested by Brownlee et al. contains a relatively small percentage of base paired residues. It is possible, however, to design other models in which a much larger number of nucleoside residues is formed into double strand helices. One such model is illustrated in a two dimensional projection in Fig. 1. The remarkable property of this hypothetical conformation is the large fraction of residues in uninterrupted double strands. The number of residues involved in base pairing varies from 82 to 98 out of a total of 120. depending on the identity of the base in position 13 and on whether G–U pairs, two single base pairs and one loop with only two residues are included. The base composition of the 5S ribosomal RNAs would permit a theoretical maximum of 112 base paired residues. In our model as much as 87 per cent of this maximum is attained. This is a much larger percentage of double strand sections than seems possible for any of the transfer RNAs of known sequence5–8.

Studies on the structure of ribosomal ribonucleic acid from normal and tumour tissues.

The dissociation properties of ribosomal RNA from liver and hepatoma were found to be dependent upon the temperature, ionic strength and cationic valency of the solvent. On increasing the temperature the pK values of the Ν1 H— C6=O and N1 = C6 — NH2 groups of nucleobases were displaced towards neutrality due to the thermal cleavage of Η-bridge linkages. Consequently, as the temperature is increased the nucleic acids bind increasingly more equivalents of acid or alkali. On forward- and back-titration hysteresis is observed in the acid pH range and the degree of hysteresis is found to decrease as the temperature is increased. Increasing the ionic strength of monovalent cations from μ=0.005 to 0.05 Na® leads to a reduced binding of equivalents of acid and alkali brought about by the displacement of pK values towards more extreme pH-values and to an increase in the stability of secondary structure. Bivalent cations exert a stronger stabilizing effect on the molecular conformation of nucleic acids than monovalent cations of equal ionic strength. The more pronounced displacement of the PK values of ionizable groups on titration from pH 7 to pH 3 may be attributed to the additional action of bivalent cations as chelate formers. Significant differences in the acid-base properties of ribosomal RNA from liver and hepatoma were observed especially at low temperatures and high ionic strength. At 5 °C and μ=0.1 NaCl less equivalents of acid and alkali are bound by hepatoma RNA than by liver RNA due to a lower degree of dissociation particularly of the N1=C6—NH2, and Ν1H — C6=0 groups.

Circular dichroism of biological macromolecules.

Circular dichroism, the unequal absorption of right and left circularly polarized light, is a manifestation of optical activity in the vicinity of absorption bands. To the experimental scientist interested in the conformation of macromolecules and in the sensitive response of optical activity to conformational alteration, it offers a relatively new and powerful means of understanding the environment of chromophoric residues. As a tool in the elucidation of electronic spectra, it should be useful to the theoretical scientist in identifying weakly allowed absorption bands as well as in providing rotational parameters which can be compared with the developing theory of optical activity. I have stressed application of circular dichroism, to experimental aspects of protein and nucleic acid conformation in solution. Much is still uncertain in particular quantitative details. However, even these early results shed new light and yield new information on the conformation of these molecules.

Studies on nucleic acid metachromasy. I. The effect of certain fixatives on the dye stacking properties of nucleic acids in solution.

The stacking coefficients (K's) of nucleic acids have been thought to influence the color contrast between DNA and RNA in tissue sections stained with metachromatic dyes. This idea was tested by titrating toluidine blue (TB) and acridine orange (AO) in solution against DNA and RNA, native or treated with formaldehyde, acrolein, or Carnoy's fluid. Absorption spectra at varying polymer-dye ratios were used to compute K values by the methods of Bradley and colleagues. Results with both dyes fit Bradley's stacking equations. Fixatives did not block dye-binding sites but markedly altered K values. K of DNA was low, unaffected by aldehyde fixative, increased by Carnoy's fluid or heat denaturation. K of RNA was higher than that of DNA and was increased greatly by formaldehyde, almost as much by acrolein, considerably less by Carnoy's fluid. Aldehyde effects were partially reversed upon removal of aldehyde by dialysis. These observations accord with known effects of aldehydes and denaturation upon nucleic acid conformation. Differences between K's of DNA and RNA were greater after aldehyde treatment than after Carnoy's, and were greater with AO than with TB. This is generally consistent with the magnitude of the color contrasts observed in tissues. Additional factors must contribute to the intense color contrast observed in acrolein-fixed tissues stained with TB.

Partition studies on nucleic acids: I. Influence of electrolytes, polymer concentration and nucleic acid conformation on the partition in the dextran-polyethylene glycol system

1. Partition of nucleic acids in liquid two-phase systems composed of two polymers, dextran and polyethylene glycol, and water is studied with respect to the addition of different electrolytes, polymer concentration and nucleic acid conformation. 2. The nucleic acids tested include native and heat denatured DNA from calf thymus, polyadenylic acid, polyuridylic acid, polycytidylic acid and polyinosinic acid. 3. Partition of nucleic acids depends highly on the electrolyte composition, both the ionic strength and the kinds of ions. A small shift in the ratio between two cations or two anions can thus transfer high-molecular-weight nucleic acids from one phase to the other. This transfer is specific for each type of nucleic acid. 4. The partition of single-stranded forms is compared with double or triple-stranded forms in systems with defined electrolyte compositions. It is demonstrated that a change from a single-stranded to double or triple-stranded forms is accompanied with an increase in the partition coefficient of 100–1000 times. 5. It is demonstrated that an 1:1 mixture of polyadenylic acid and polyinosinic acid results in the formation of a mixture of a three-stranded complex poly (adenylic acid + 2 inosinic acids) plus polyinosinic acid.

A study of conformation of nucleic acids in solution by means of circular dichroism

The circular dichroism of various ribo- and deoxyribonucleic acids was measured in the spectral region of 340 to 230 m μ ; all ribonucleic acids (transfer RNA, ribosomal RNA, tobacco mosaic virus RNA and Reovirus RNA) exhibit similar curves composed of a positive band centred at 265 m μ . The intensity of this band gradually decreases in conditions favouring helix-coil transitions, at higher temperatures or at increasing concentrations of ethanol, parallel to the increase in absorbance. This identity of the curves for circular dichroism of various RNA's indicates that within all native RNA's an important fraction of the molecule must have a comparable secondary structure. The circular dichroic spectra of DNA from calf thymus, fowl erythrocytes and Escherichia coli also lead to identical results. The curve is composed of two parts: one positive, with a maximum at 273 m μ ; and one negative, with a minimum at 243 m μ . At high temperature a decrease of intensity of circular dichroism was observed, but at intermediate temperatures (about 40 to 55°C) the intensity of the positive band increases without any observable changes in absorbancy. In 80% alcohol at low salt concentration, the circular dichroic curve of DNA is essentially composed of a positive band, the rotational strength of which is about three times greater than that of the native form. The spectra of DNA in alcohol compared to native DNA in aqueous solutions indicate an increased absorption in the region of 275 m μ . It is suggested that DNA in 80% alcohol exists in an intermediate form which is different from its native and random coil conformations. In contrast to polyriboadenylic acid, the intensity of the positive circular dichroic bands of nucleic acids, DNA and RNA, are relatively low; this probably results from certain geometrical features of their helical structures.