Mechanisms of chain folding in nucleic acids. The (omega, omega) plot and its correlation to the nucleotide geometry in yeast tRNAPhe1.

The (omega', omega) polot depicting the internucleotide P-O bond rotation angles in yeast phenylalanyl transfer RNA has established the interdependence of the phosphodiesters and the nucleotide geometries in the folding of the polynucleotide backbone. The plot distinguishes the regions characteristic of secondary helical structures and tertiary structural loops and bends. The folding of the polynucleotide chain is accomplished either solely by rotations around the P-O bonds or in concert with rotations around the nucleotide C4'-C5' bond with or without changes in the sugar ring pucker. In spite of differences in nucleotide sequence and intraloop tertiary interactions in the anticodon and pseudouridine loops, a characteristic repeating structural unit is found for the sugar-phosphate backbone of the tetranucleotide segment around the sharp turns.     

Configuration-dependent properties of randomly coiling polynucleotide chains. I. A comparison of theoretical energy estimates

Various theoretical estimates of the conformational energy associated with polynucleotides in solution have been compared with each other and also with the experimentally observed conformations found in X-ray crystallographic investigations of low-molecular-weight nucleic acid analogs. In view of the disparities between these data, certain configuration-dependent properties (i.e., the mean-square unperturbed end-to-end distance 〈r²〉₀ and the average vicinal nmr coupling constant 〈J〉) appropriate to randomly coiling polynucleotides described by either the energy estimates or by the crystallographically preferred conformations have also been calculated and compared with the known solution behavior of polynucleotide chains. Both the theoretical energy surfaces and the X-ray data show good agreement with the nmr coupling constant indications of the preferred rotations about the O-C and C-C bonds of the chain backbone. The principal discrepancies between the theoretical methods and X-ray data arise in their ability to predict successfully the preferred rotations about the two phosphodiester bonds of the chain backbone and the unperturbed dimensions of randomly coiling polynucleotide chains.     

Random coil conformations of polynucleotides: a study incorporating long-range correlations due to dynamics of the sugar residue

A novel virtual bond scheme joining the atoms P-C4′ and C4′-P of a nucleotide repeat, consistent with the stereochemistry of polynucleotide chains, has been developed. The scheme, with its inherent feature to account for all the major sources of flexibility, could also consider effectively the short range and long range interactions, thereby simplifying the analysis of random coil and ordered structures. Using this scheme, unperturbed end-to-end dimensions and persistence lengths of polynucleotide chains have been computed incorporating the dynamical aspects of the sugar ring as well as the C4′ C5′ bond and the correlated changes in phosphodiester conformations. Calculated unperturbed dimensions are in excellent agreement with experimental values. Results show that the random coil is characterized by a large proportion of helical segments of the A-form.     

On molecular mechanisms of nucleic acid synthesis fidelity aspects. 1. Contribution of base interactions

This paper is concerned with the molecular mechanisms of spontaneous replacements of base pairs in the processes of template synthesis of nucleic acids. The method of atom-atom potential functions was used to calculate the energies of interaction in non-complementary base pairs arranged in a common plane so that the mutual position of the glycosidic bonds does not differ appreciably from their position for Watson-Crick pairs in the DNA double helix. A number of local minima of this energy have been found, which could occur in template synthesis and result in insertion of incorrect bases into the double helix. The calculation results are indicative of formation of purine-purine pairs with one of the purine nucleotides in syn-conformation, which can be regarded as a typical pathway of transversion, and that of wobble-pairs TG and AC, which can be regarded as a typical pathway of transition. The contribution of intramolecular interactions of nucleic acids as well as interactions of polynucleotide chains with an enzyme to the fidelity of template synthesis of nucleic acids is discussed. The calculation results are compared with the experimental data on the frequency of spontaneous mutations and the frequency of errors in template synthesis of nucleic acids in vitro.     

The flexible DNA double helix. II. Superhelix formation.

A simple super or s-virtual bond scheme has been developed for the treatment of tertiary or superhelical structure in polynucleotide chains. The various spatial configurations accessible to the flexible double helix are rendered more readily intelligible by the introduction of these hypothetical bonds to replace real sequences of regular secondary structure. The scheme is utilized to examine the enormous variety of tertiary structure that can be generated by regularly bending a B-DNA reference helix at the phosphodiester linkages. Of particular interest from the study are the large families of bends that generate superhelices of identical macroscopic dimensions. Various modes of folding the B-type helix into superhelices that fit the experimentally measured dimensions of chromatin nucleosomes are illustrated.     

The spatial configuration of ordered polynucleotide chains. II. The poly(rA) helix.

Approximate details of the spatial configuration of the ordered single-stranded poly(rA) molecule in dilute solution have been obtained in a combined theoretical analysis of base stacking and chain flexibility. Only those regularly repeating structures which fulfill the criterion of conformational flexibility (based upon all available experimental and theoretical evidence of preferred bond rotations) and which also exhibit the right-handed base stacking pattern observed in nmr investigations of poly(rA) are deemed suitable single-stranded helices. In addition, the helical geometry of the stacked structures is required to be consistent with the experimentally observed dimensions of both completely ordered and partially ordered poly(rA) chains. Only a single category of poly(rA) helices (very similar in all conformational details to the individual chains of the poly(rA) double-stranded X-ray structure) is thus obtained. Other conformationally feasible polynucleotide helices characterized simply by a parallel and overlapping base stacking arrangement are also discussed.     

Polynucleotide folding in yeast tRNAPhe: elucidation of short-, medium-, and long-range interactions of sugar-phosphate-sugar backbone and base using a "blocked" nucleotide probe

It has recently been proposed that the repeating backbone nucleotide may be regarded as consisting of two blocks of equal magnitude representable by two virtual bonds. Implicit consideration of the nucleotide (ψ,ψ) and internucleotide (ω′,ω) geometry that generate variety in polynucleotide conformations, and of the constancy of the repeating structural moieties (P-C4′ and C4′-P) independent of the above rotations, has enabled us to utilize this scheme in the study of ordered structures such as di-, oligonucleotides and, most significantly, tRNA. The polynucleotide folding dictated by short-, intermediate-, and long-range interactions in the monoclinic and orthorhombic forms is described and compared through circular plots depicting the virtual bond torsions and distance plots constructed independently for backbone as well as bases. The torsions and the bond angles associated with the virtual bonds afford a clear distinction between ordered helical segments from loops and bends of tRNA. Lower virtual bond torsions (?60° to 60°) concomitant with higher values of virtual bond angles characterize various bend regions, while torsions around 160°–210° typify ordered helical strands. The distance plot elucidates the type of interaction associated with various sub-structures (helix–helix, helix–loop, and loop–loop) that form the constituents of different structural domains. Several other features such as the manifestation of the P₁₀ loop and the approximate twofold symmetry in the tRNA molecule are conspicuous on the distance plot.     

The spatial distributions of randomly coiling polynucleotides

Statistical mechanical averages of vectors and tensors characterizing the allowed configurations of randomly coiling polynucleotides have been calculated for chains of 2⁰–2¹⁰ repeating units. Specifically, the persistence vector p = 〈 r 〉 has been obtained as a function of chain length. Configurational averages of the Cartesian tensors formed from the displacement vector ρ = r – p have been computed up to and including the tensor of seventh rank. From these tensors the three-dimensional spatial distributions of end-to-end vectors have been constructed to provide comprehensive pictures of the directional tendencies of the randomly coiling polynucleotide. The elements of the third and fourth moment tensors, however, give sufficient information to represent accurately the qualitative features of the spatial distributions. For long chains, more than 2⁶ (64) repeating units, the spatial distributions assume spherically symmetric shapes that can be adequately characterized by one-dimensional radial distribution functions. These radial distribution functions agree well with the radial distributions calculated from Monte Carlo samples containing more than 5000 chains. The constraints of fixed bond lengths, fixed bond angles, and hindered internal rotations severely distort the spatial distributions of short polynucleotide chains to mushroom-shaped volumes. These skewed distributions provide information useful to the analysis of small, single-stranded loops, bulges, and circles. The formation of these structures requires the termini of the polynucleotides to lie within specifically delineated volumes with respect to coordinate systems affixed to the first bonds of the chains. The extent to which these loop closure volumes overlap the three-dimensional spatial distributions provides estimates of loop formation that are much more reliable than earlier studies based upon the radial distribution function.     

Noninteger pitch and nuclease sensitivity of chromatin DNA.

Assuming that variation of nuclease sensitivity along nucleosomal DNA can basically be attributed to orientations of sugar--phosphate bonds relative to histone core, the pitch of chromatin DNA is estimated to be 10.33--10.40 base pairs. This is in accordance both with the known measured average distance between cleavage sites (10.3--10.4 base pairs) and with published data on variation of relative sensitivities of these sites to nuclease attack. The variation can be explained solely as a result of the systematic change of orientation of sugar--phosphate bonds of sensitive sites without additional suggestions about local steric hindrances by histone molecules. According to the analysis locations of sites least sensitive to nuclease attack should not depend on kind of endonuclease though the stagger could differ. We conclude that the nucleosome core particle is axially symmetrical. The results strongly support the suggestion that DNA is wrapped around the histone octamer smoothly, without interruption of base-stacking interactions.     

Side-chain flexibility in protein-ligand binding: the minimal rotation hypothesis

The goal of this work is to learn from nature about the magnitudes of side-chain motions that occur when proteins bind small organic molecules, and model these motions to improve the prediction of protein-ligand complexes. Following analysis of protein side-chain motions upon ligand binding in 63 complexes, we tested the ability of the docking tool SLIDE to model these motions without being restricted to rotameric transitions or deciding which side chains should be considered as flexible. The model tested is that side-chain conformational changes involving more atoms or larger rotations are likely to be more costly and less prevalent than small motions due to energy barriers between rotamers and the potential of large motions to cause new steric clashes. Accordingly, SLIDE adjusts the protein and ligand side groups as little as necessary to achieve steric complementarity. We tested the hypothesis that small motions are sufficient to achieve good dockings using 63 ligands and the apo structures of 20 different proteins and compared SLIDE side-chain rotations to those experimentally observed. None of these proteins undergoes major main-chain conformational change upon ligand binding, ensuring that side-chain flexibility modeling is not required to compensate for main-chain motions. Although more frugal in the number of side-chain rotations performed, this model substantially mimics the experimentally observed motions. Most side chains do not shift to a new rotamer, and small motions are both necessary and sufficient to predict the correct binding orientation and most protein-ligand interactions for the 20 proteins analyzed.     

Simulation of double-stranded branch point migration.

A structural and dynamic model has been developed for the branch point formed when two DNA double helices exchange strands during genetic recombination. This model, which generalizes most previous structural models, maintains the twofold symmetry inherent in the covalent and hydrogen bonded structure, yet has three degrees of freedom about virtual bonds, constituting a simplified junction. Using this structural model, a three-step dynamic model for branch point migration has been developed: longitudinal diffusion about the virtual bonds to achieve a structure in which the helix axes are approximately parallel; opening of the base pairs; and rotary diffusion about the helix axis to effect a migratory event. The model, which includes the possible role of electrostatic interactions, solves problems inherent in previous treatments. We find that no significant electrostatic torques arise that promote branch point migration. The absence of a kinetic mechanism to circumvent thermodynamic barriers due to mispairing suggests that an energy source is used for those situations in living systems.     

Determinants of strand register in antiparallel beta-sheets of proteins.

Antiparallel beta-sheets present two distinct environments to inter-strand residue pairs: beta(A,HB) sites have two backbone hydrogen bonds; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only approximately 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic-Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi1 conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.     

Syn-anti effects on the spatial configuration of polynucleotide chains

Semiempirical energy calculations have beeb performed on model nucleic acid systems to assess the preferred conformation of the rotation χ about the glycosidic linkage and also the effect of this rotation on the spatial configuration of the sugar-phosphate chain backbone. The rotation angle ?? about bond C_5′–C_4′ in purine polyribonucleotides and 5′-monoribonucleotides is found to depend on whether the conformational range of χ is syn or anti. The preferred conformation of χ in these molecules is also found to depend upon the nature of the attached base. The orientation of χ in poly rA chains is predicted to be predominantly anti, whereas in poly rG the syn conformer is expected to occur in significant proportions. The syn conformer is preferred almost exclusively in certain unusual purine polynucleotides, such as poly 8Br-rA. It is noted that the preferred conformation of x in polynucleotides is not necessarily the same as that calculated for 5′-mononucleotides and nucleosides. On the basis of these calculations, the influence of the orientation and nature of a purine base on the spatial configuration of a polynucleotide chain as a whole has been examined. The random coil dimensions of a syn polynucleotide chain are found to be larger than those of an anti chain as a consequence of the effect of a syn base on the local conformation of the chain skeleton. Finally, it is found that the occurrence of a syn base in an ordered polynucleotide chain may prevent the formation of normal stacking with the preceding base.     

Computational studies of polynucleotide flexibility.

Details of polynucleotide flexibility may be probed through a combination of semiempirical potential energy calculations and statistical mechanical analyses. The pseudorotational motions of the furanose and the long-range correlated rotations of the chain backbone are described briefly here.     

Theory of the influence of oligonucleotide chain conformation on double helix stability

We consider theoretical aspects of reactions that form base pairs in a double helix. The equilibrium constant for such reactions depends on the probability of finding the two bases in the correct orientation for pairing. This probability can be expressed in terms of the spatial and angular distribution of one micleotide around the other. In this paper we use Monte-Carlo techniques to calculate the distribution of distances between chosen phosphates in nonhclical oligonucleotide backbones, using crystallographic data for bond lengths and angles, and a screened Coulomb potential for phosphate–phosphate interactions. The model chosen is one that predicts correctly the observed dimensions of an unperturbed polynucleotide chain. Knowledge of distance distribution functions permits calculation of the dependence on loop size of the probability of closing a single backbone strand into a hairpin helix. Our results agree roughly, although not exactly, with the semiempirical ring-weighting functions determined by Schefller. Elson, and Baldwin. Further results are a comparison of intramolecular and bimolecular helix nucleation equilibrium constants and a calculation of the stacking free energy in a double helix.     

The spatial configuration of ordered polynucleotide chains. I. Helix formation and base stacking.

A single virtual bond scheme set forth previously for the treatment of average properties of randomly coiling polynucleotides is here applied to the calculation of helical parameters which characterize a regularly repeating polynucleotide molecule. Only a fraction of the enormous number of conformationally feasible helixes fulfill the geometric criteria of vertical base stacking usually associated with ordered polynucleotide chains. Detailed examination of the nature and mode of base stacking feasible in a single helical backbone structure indicates that the handedness of a base stacking arrangement does not correlate either quantitatively or qualitatively with the handedness of the polymer backbone. A number of polynucleotide chains which exhibit lefthanded base stacking patterns in nmr and CD studies may, in fact, be righthanded helixes.     

Crystal structure of the topoisomerase II poison 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide bound to the DNA hexanucleotide d(CGTACG)2.

The structure of the complex formed between d(CGTACG)(2) and the antitumor agent 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide has been solved to a resolution of 1.6 A using X-ray crystallography. The complex crystallized in space group P6(4) with unit cell dimensions a = b = 30.2 A and c = 39.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The asymmetric unit contains a single strand of DNA, 1. 5 drug molecules, and 29 water molecules. The final structure has an overall R factor of 19.3%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and the protonated dimethylamino group partially occupies positions close to ( approximately 3.0 A) the N7 and O6 atoms of guanine G2. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Sugar rings adopt the C2'-endo conformation except for cytosine C1 which moves to C3'-endo, thereby preventing steric collision between its C2' methylene group and the intercalated acridine ring. The intercalation cavity is opened by rotations of the main chain torsion angles alpha and gamma at guanines G2 and G6. Intercalation perturbs helix winding throughout the hexanucleotide compared to B-DNA, steps 1 and 2 being unwound by 8 degrees and 12 degrees, respectively, whereas the central TpA step is overwound by 17 degrees. An additional drug molecule, lying with the 2-fold axis in the plane of the acridine ring, is located at the end of each DNA helix, linking it to the next duplex to form a continuously stacked structure. The protonated N,N-dimethylamino group of this "end-stacked" drug hydrogen bonds to the N7 atom of guanine G6. In both drug molecules, the 4-carboxamide group is internally hydrogen bonded to the protonated N-10 atom of the acridine ring. The structure of the intercalated complex enables a rationalization of the known structure-activity relationships for inhibition of topoisomerase II activity, cytotoxicity, and DNA-binding kinetics for 9-aminoacridine-4-carboxamides.     

Correlation between the backbone and side chain conformations in 5′-nucleotides. The concept of a ‘rigid’ nucleotide conformation

Conformational energies of the 5′-adenosine monophosphate have been computed as a function of χ and ψ, of the torsion angles about the side-chain glycosyl C(1′)–N(9) and of the main-chain exocyclic C(4′)–C(5′) bonds by considering nonbonded, torsion, and electrostatic interactions. The two primary modes of sugar puckering, namely, C(2′)-endo and C(3′)-endo have been considered. The results indicate that there is a striking correlation between the conformations about the side-chain glyocsyl bond and the backbone C(4′)–C(5′) bond of the nucleotide unit. It is found that the anti and the Gauche–Gauche (gg), conformations about the glycosyl and the C(4′)–C(5′) bonds, respectively, are energetically the most favored conformations for 5′-adenine nucleotide irrespective of whether the puckering of the ribose is C(2′)-endo or C(3′)-endo. Calculations have also shown that the other common 5′-pyrimidine nucleotides will show similar preferences for the glycosyl and C(4′)–C(5′) bond conformations. These results are in remarkable agreement with the concept of the “rigid” nucleotide unit that has been developed from available data on mononucleotides and dinucleoside monophosphates. It is found that the conformational ‘rigidity’ in 5′-nucleotides compared with that of nucleosides is a consequence of, predominantly, the coulombic interactions between the negatively charged phosphate group and the base. The above result permits one to consider polynucleotide conformations in terms of a “rigid” C(2′)-endo or C(3′)-endo nucleotide unit with the major conformational changes being brought about by rotations about the P–O bonds linking the internucleotide phosphorus atom. IT is predicted that the anti and the gg conformations about the glycosyl and the C(4′)–C(5′) bonds would be strongly preferred in the mononucleotide components of different purine and pyrimidine coenzymes and also in the nucleotide phosphates like adenodine di- and triphosphates.     

Total synthesis of a structural gene for the human peptide hormone angiotensin II.

Seven oligonucleotide chains containing between 6 and 11 nucleotide units were synthesized. The segments were phosphorylated by T4 polynucleotide 5'-hydroxyl-kinase and joined by T4 polynucleotide synthetase (ATP) to give the double-stranded DNA consisting of 33 base pairs. The DNA sequence was deduced from the known peptide sequence according to the genetic code.