Variability in the geometry of RNA double helices generated from dinucleoside building-blocks

A graphical method is presented for the generation of helical parameters from single-crystal structures of RNA nucleic acid fragments that are minimally dinucleosides. The method is compared with other published procedures, for a number of text examples. The RNA double helices generated from three different salts of the dinucleoside monophosphate GpC are examined in relation to the variations in helix morphology that are produced. It is shown that small differences between these GpC salts can be amplified to very distinct helix characteristics.     

Filamentous bacterial viruses. XIL. Molecular architecture of the class I (fd, If1, IKe) virion

The X-ray diffraction patterns of the fd, If1 and IKe strains of filamentous bacterial viruses (class I) indicate that the arrangement of capsid proteins in the virion approximates a left-handed helix of 15 Å pitch with 4.5 units per turn. The protein molecules are each elongated in an axial direction, and also slope radially, so as to overlap each other and give an arrangement of molecules reminiscent of scales on a fish. This helix of capsid proteins is related to the class II helix by a small twist about the helix axis. The protein molecules are also perturbed (by a few Ångström units) away from the positions that they would occupy in a simple 4.5 units per turn helix. The perturbation repeats about every five protein molecules, and is mainly axial. This arrangement of proteins forms a tube with inner diameter about 20 Å and outer diameter about 60 Å, encapsulating the DNA.     

Computer building of β-helical polypeptide models

A general method is presented for computing the atomic coordinates of helices in which a dipeptide is the repeating unit. The method will generate both single- and double-stranded model helices having idealized bond lengths and angles, and any arbitrary, user-specified, pitch and number of residues per turn. The variation of inter- and intrastrand hydrogen bonds with pitch and number of residues per turn can thus be examined. An application of the method is the construction of a β-helix having pitch of 6.3 Å per turn and 4.85 residues per turn, a model which can pack nicely into the unit cell of crystals of cation-bound gramicidin A.     

Parallel stranded DNA under scanning tunnelling microscope: the main characteristics of the double helix.

Using the scanning tunnelling microscopy we have directly observed the parallel stranded DNA of 43 bp in length, containing alternating AT-stretches. The double helix is right-handed and has the same width of each grooves equal to 17.4 A. The average pitch of the helical turn is about 34 A. The parallel double helix possesses no more than 8.6 bases per one turn. The diameter of the parallel stranded DNA molecule is 17-18 A. We conclude that in parallel DNA double helix the angle between N-glycoside bounds in trans-Crick-Watson base pairs is close to 180 degrees.     

A structural model for the polyadenylic acid single helix.

In the crystal, the poly(A) fragment ApApA assumes a conformation with the 5′-terminal and middle adenosines in a single helical arrangement. From the atomic co-ordinates of these two nucleotides the structure of the poly(A) single helix was derived mathematically. The helix has a pitch height of 25.4 Å, nine nucleotides per turn and the normals to the adenine bases form an angle of 66 ° with the helix axis.     

Conformational stability in dinucleoside phosphate crystals. Semiempirical potential energy calculations for uridylyl-3'-5'-adenosine monophosphate (UpA) and guanylyl-3',5'-cytidine monophosphate (GpC)

Classical potential energy calculations were performed for the dinucleoside phosphates UpA and GpC. Two widely accessible low-energy regions of conformation space were found for the ω′, ω pair. That of lowest energy contains conformations similar to helical RNA, with ω′ and ω in the vicinity of 300° and 280°, respectively. All five experimental observations of crystalline GpC, two of ApU, and the helical fragment of ApApA fall in this range. The second lowest region has ω′ and ω at about 20° and 80°, respectively, which is in the general region of one experimentally observed crystalline conformer of UpA, and the nonhelical region of ApApA. It is concluded that GpC and ApU, which were crystallized as either sodium or calcium salts, are shielded from each other in the crystal by the water of hydration and are therefore free to adopt their predicted in vacuo minimum energy helical conformations. By contrast, crystalline UpA had only 1/2 water per molecule, and was forced into higher energy conformations in order to maximize intermolecular hydrogen bonding.     

ω-Helices in Proteins

A modification of the α-helix, termed the ω-helix, has four residues in one turn of a helix. We searched the ω-helix in proteins by the HELFIT program which determines the helical parameters—pitch, residues per turn, radius, and handedness—and p = rmsd/(N ? 1)^1/2 estimating helical regularity, where “rmsd” is the root mean square deviation from the best fit helix and “N” is helix length. A total of 1,496 regular α-helices 6–9 residues long with p ≤ 0.10 Å were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical α-helix and ω-helix. Sixty-two right handed ω-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the ω-helices occur really in proteins.     

Variability in the Geometry of RNA Double Helices Generated from Dinucleoside Building-Blocks

Abstract

A graphical method is presented for the generation of helical parameters from single-crystal structures of RNA nucleic acid fragments that are minimally dinucleosides. The method is compared with other published procedures, for a number of text examples. The RNA double helices generated from three different salts of the dinucleoside monophosphate GpC are examined in relation to the variations in helix morphology that are produced. It is shown that small differences between these GpC salts can be amplified to very distinct helix characteristics.     

RNA double-helical fragments at atomic resolution: II. The crystal structure of sodium guanylyl-3′,5′-cytidine nonahydrate

The crystal structure of sodium guanylyl-3′,5′-cytidine (GpC) nonahydrate has been determined by X-ray diffraction procedures and refined to an R value of 0.054. GpC crystallizes with four molecules per monoclinic unit cell, space group C2, with cell dimensions: $\text{a = 21.460, b = 16.297, c = 9.332}\text{A}\text{?}\text{and}\text{β = 90.54 °}$. Two molecules of GpC related by the 2-fold axis of the crystal form a small segment of right-handed, anti-parallel double-helical RNA in the crystal. Guanine is paired to cytosine through three hydrogen bonds of lengths 2.91, 2.95 and 2.86 Å. The bases along each strand are heavily stacked at a distance of about 3.4 Å. The fragments form skewed flattened rods within the lattice by the inter-molecular stacking of guanines with each other and the stacking of cytosine with the guanosine Ol′atom. The sodium cations are bound only to the ionized phosphate groups in this structure and exhibit face-sharing octahedral co-ordination. The sodium cations serve to bridge the rods of GpC fragments and organize them into sheets within the crystal. There are 18 water molecules per double-helical fragment which are all part of the first co-ordination shell of nitrogen, oxygen or sodium atoms.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Geometry of experimentally observed RNA residues in tRNA and dinucleoside monophosphates: the effect of small variations in the backbone angles.

The polymerization of various experimentally observed conformers of RNA from tRNA and some dinucleoside monophosphates have been examined with a program that computes the basic helix parameters directly from the six backbone torsion angles ω′, ?′, ψ′, ψ, ?, ω to give n (= 360/θ), the number of residues per turn; h, the rise per residue; and r, the radius of the phosphate atoms from the helix axis. The single-stranded regions of tRNA that have A-form residues have a notably lower value of n than the double-stranded regions. The G-U “wobble” base pair is shown to be an energetically strained left-handed form. The A-form dinucleoside monophosphates also have a low value of n. A model of UpAl polymerized as a fourfold left-handed helix with the bases on the outside and phosphates on the inside is investigated for its sharp 90° turn angle characteristics. UpA2 cannot be polymerized due to a low values of h (1.31 Å) and r (2.72 Å), which cause steric hindering. An eightfold model of poly(rA) is discussed as are the nonhelical residues of tRNA. Finally, the effects of small changes in dihedral angles and bond lengths and angles on the helical parameters are investigated and discussed by way of explaining this behavior.     

Changes in the pitch of the myosin helix preceding cross-brdige attachment in oscillatory contractions of frog sartorius muscle

When glycerinated fibres in presence of 5·10^?7M Ca²⁺ were oscillated at a frequency of 10 Hz, the X-ray intensities at 429 Å and at the small radial position of the 191 Å layer-line change in an inverse pattern. This rearrangement of the pitch of the myosin helix precedes by at least 10 msec the intensity increase on the 59 Å X-ray layer-line, which provides a measure for cross-bridge attachment to the actin. The perfect match of the new pitch of the myosin helix with the pitch of the actin helix may ensure optimal cross-bridge attachment.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

High salt solution structure of a left-handed RNA double helix

Right-handed RNA duplexes of (CG)_n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)₂ determined in 6 M NaClO₄. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6_(n)-H5′/H5″_(n)-H8_(n+1)-H1′_(n+1)-H6_(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4–7°), whereas GpC steps have large negative values (−61°). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2′-OH groups, and the major groove are well defined. The 2′-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2′-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.     

Spatial structure of gramicidin A in organic solvents. 1H-NMR analysis of conformation heterogeneity in ethanol

Structural features of double helices formed by polypeptides with alternating L- and D-amino acid residues were analysed. It was found that the map of short distances (less than 4 A) between protons of the two backbones is unique for each double helix type and even its fragment implies unambiguously parameters of the helix (i.e. parallel or antiparallel, handedness, pitch of helix, relative shift of polypeptide chains). By analysis of two-dimensional 1H-NMR spectra (COSY, RELSY, HOHAHA, NOESY), proton resonances of [Val1]gramicidin A (GA) in the ethanol solution were assigned. The results obtained show that the solution contains five stable conformations of GA in comparable concentrations. Monomer of GA is in a random coil conformation. Specific maps of short interproton distances for the other four species (1-4) were obtained by means of two dimensional nuclear Overhauser effect spectroscopy. The maps as well as spin-spin couplings of the H-NC alpha-H protons and solvent accessibilities of the individual amide groups correspond to four types of double helices pi pi LD 5,6 with 5.6 residues per turn. The double helices are related to the Veatch species 1-4 of GA. Species 1 and 2 are left-handed parallel double helices increase increase pi pi LD 5,6 with different relative shift of polypeptide chains. Species 3 is a left-handed antiparallel double helix increase decrease pi pi LD 5,6 and species 4 is a right-handed parallel double helix increase increase LD 5,6. In the dimers helices are fixed by the maximum number (28) of interbackbone hydrogen bonds NH...O = C possible for these structures. Species 1, 3 and 4 have C2 symmetry axes. Relationship between gramicidin A spatial structures induced by various media is discussed.     

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance.

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.     

RNA sequences specifically associated with mouse intracisternal A particles.

Electron microscopic examination of the histone H1-depleted, folded genomes of Drosophila melanogaster reveals that they are composed of long cylindrical cables of about 100 Å diameter. Limited single-strand nicking with DNAase I relaxes the 100 Å fibers to a “beads-on-a-string” structure, showing the nucleosomes and internucleosome DNA.Based on these results and other available data, we have constructed a detailed space-filling model for the higher order DNA coiling in chromatin, starting with the symmetrical nucleosome core previously described (Weintraub, Worcel and Alberts, 1976). The model defines the path of the DNA helix and the nucleosome arrangement along the DNA coil for both the 100 Å and the 200–300 Å fibers.Following Sobell et al. (1976), we believe that the DNA is coiled in the 100 Å nucleofilament in a uniform left-handed supercoil of about 90 base pairs (bp) per turn and 47 Å pitch; the 140 bp symmetrical nucleosome cores align themselves along this uniform DNA superhelix so that the isologous outer surfaces of adjacent nucleosomes touch and the internucleosome spacer DNA coils between them. A few single-strand discontinuities [about one nick per 85 kilobases (kb); Benyajati and Worcel, 1976] in the H1-depleted 100 Å fiber can thus relax the negatively supercoiled internucleosome DNA generating the “beads-on -a-string” appearance.We propose that histone H1 binds to the 100 Å diameter superhelix and coils it into tightly packed, 110 Å pitch super-superhelices (“solenoids;” Finch and Klug, 1976) of variable diameter (between 200–300 Å). In our model, the “thick” 200–300 Å fiber is stabilized at metaphase by histone H1-H1 heterologous interactions between adjacent helical turns of the nucleofilament, and the internucleosome spacer DNA is located on the outside. Symmetry considerations demand that changes in the length of the repeat should lead to variations in the number of nucleosomes per helical turn and in the handedness of these turns in the 200–300 Å metaphase fiber.     

Characterization at atomic resolution of peptide helical structures.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.