Transcription of the his3 gene region in Saccharomyces cerevisiae

The dodecamer d(CpGpCpGpApApTpTpCpGpCpG) or C-G-C-G-A-A-T-T-C-G-C-G crystallizes as slightly more than one full turn of right-handed B-DNA. It is surrounded in the crystal by one bound spermine molecule and 72 ordered water molecules, most of which associate with polar N and O atoms at the exposed edges of base-pairs. Hydration within the major groove is principally confined to a monolayer of water molecules associated with exposed N and O groups on the bases, with most association being monodentate. Waters hydrating backbone phosphate oxygens tend not to be ordered, except where they are immobilized by 5-methyl groups from nearby thymines. In contrast, the minor groove is hydrated in an extensive and regular manner, with a zigzag “spine” of first- and second-shell hydration along the floor of the groove serving as a foundation for less-regular outer shells extending beyond the radius of the phosphate backbone. This spine network bridges purine N-3 and pyrimidine O-2 atoms in adjacent base-pairs. It is particularly regular in the A-A-T-T center, and is disrupted at the C-G-C-G ends, in part by the presence of the N-2 amino groups on guanine residues. The minor groove hydration spine may be responsible for the stability of the B form of polymers containing only A · T and I · C base-pairs, and its disruption may explain the ease of transition to the A form of polymers with G · C pairs.     

Ordered transcription of RNA tumor virus genomes.

The crystal structure of sodium adenylyl-3′,5′-uridine (ApU) hexahydrate has been determined by X-ray diffraction procedures and refined to an R factor of 0.057. ApU crystallizes with two molecules per asymmetric unit in a monoclinic unit cell, space group P2₁, with cell dimensions: $\text{a = 18.025, b = 17.501, c = 9.677}\text{A}\text{?}\text{and}\text{β = 99.45 °}$. The two independent molecules of ApU form a small segment of right-handed antiparallel double-helical RNA in the crystal, with Watson-Crick base-pairing between adenine and uracil. This is the first time that this Watson-Crick base-pair has been seen unambiguously at atomic resolution and it is also the first time that a nucleic acid fragment with double-helical symmetry has been seen at atomic resolution. The distance between the C1′ atoma of the adenine-uracil base-pair is slightly shorter than the analogous distance seen in guanine-cytosine base-pairs. The bases in each strand are heavily stacked. One sodium cation binds to the phosphates, as expected; however, the other sodium cation binds on the dyad axis in the minor groove of the double helix. It is co-ordinated directly to the two uracil carbonyl groups which protrude into the minor groove and is shielded from the nearest phosphates by a shell of water. This binding appears to be sequence-specific for ApU. One of the adenines also forms a pair of hydrogen bonds to a nearby ribose, utilizing N6 and N7. The 12 water molecules per double-helical fragment are all part of the first co-ordination shell. The ions and the symmetry of the double-helical fragment are the major organizing elements of the solvent region.     

Amide protein exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution. Studies with two-dimensional nuclear magnetic resonance

Dickerson and his colleagues have described the structure of the DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G in the B form at a level that shows clearly several aspects of some base sequence-dependent departures from the ideal, regular helical structure of B-DNA. I argue that the detailed conformation is a consequence of simple steric repulsive forces between purine bases in consecutive base-pairs but on opposite backbones. These repulsions are a consequence of the “propeller twist” of the base-pairs, together with the larger size of the purine bases, and they may occur in either the major or the minor groove. The argument is conducted in terms of the structural mechanics of a deformable elastic system. These repulsive forces between the base-pairs are resisted by stresses in the helical backbones, which may be studied quantitatively via the variation in torsion angles δ along the backbones, at the points where the sugar rings are connected. There is also a correlation between the cross-chain purine repulsions and the perturbations in helical twist angle between successive base-pairs. The work suggests some comments on the proposed “alternating B” form, the Z form and the A form of DNA.     

Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure

Detailed examination of the structure of the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, obtained by single-crystal X-ray analysis (Drew et al., 1981), reveals that the local helix parameters, twist, tilt and roll, are much more strongly influenced by base sequence than by crystal packing or any other external forces. The central EcoRI restriction endonuclease recognition site, G-A-A-T-T-C, is a B helix with an average of 9.8 base-pairs per turn. It is flanked on either side by single-base-pair steps having aspects of an A-like helix character. The dodecamer structure suggests several general principles, whose validity must be tested by other B-DNA analyses. (1) When an external bending moment is applied to a B-DNA double helix, it bends smoothly, without kinks or breaks, and with relatively little effect on local helix parameters. (2) Purine-3′,5′-pyrimidine steps open their base planes towards the major groove, pyrimidine-purine steps open toward the minor groove, and homopolymer (Pur-Pur, Pyr-Pyr) steps resist rolling in either direction. This behavior is related to the preference of pyrimidines for more negative glycosyl torsion angles. (3) CpG steps have smaller helical twist angles than do GpC, as though in compensation for their smaller intrinsic base overlap. Data on A-T steps are insufficient for generalization. (4) G.C base-pairs have smaller propellor twist than A · T, and this arises mainly from interstrand base overlap rather than the presence of the third hydrogen bond. (5) DNAase I cuts preferentially at positions of high helical twist, perhaps because of increased exposure of the backbone to attack. The correlation of the digestion patterns in solution and helical twist in the crystal argues for the essential identity of the helix structure in the two environments. (6) In the two places where the sequence TpCpG occurs, the C slips from under T in order to stack more efficiently over G. At the paired bases of this CpG step, the G and C are tilted so the angle between base planes is splayed out to the outside of the helix. This TpC is the most favored cutting site for DNAase I by a factor of 4.5 (Lomonossoff et al., 1981). (7) The EcoRI restriction endonuclease and methylase both appear to prefer a cutting site of the type purine-purine-A-T-T-pyrimidine, involving two adjacent homopolymer triplets, and this may be a consequence of the relative stiffness of homopolymer base-stacking observed in the dodecamer.     

Structures for the polynucleotide complexes poly(dA) · poly(dT) and poly(dT) · poly(dA) · poly(dT)

X-ray diffraction analyses of fibers of polydeoxyadenylic acid · polydeoxythymidylic acid show that this molecule exists as a 10-fold double-helix with axial rise per nucleotide $\text{h = 3.24}\text{to}\text{3.29}\text{A}\text{?}$. The structure is very similar to B-DNA ($\text{h = 3.37}\text{A}\text{?}$) in having C3-exo furanose rings and base-pairs positioned centrally on the helix axis, but distinctive enough to have two packing modes, neither of which has been observed for B-DNA. Although the triple-stranded poly(dT) · poly(dA) · poly(dT) also has a large value of h(3.26 Å), each of the chains is a 12-fold helix of the A-genus with C3-endo furanose rings and bases displaced several Angstrom units from the helix axis.     

Higher-order structure in the 3′-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus

An experimental approach was used to determine, and compare, the higher-order structure within domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus. This domain, which encompasses approximately 300 nucleotides at the 3′ end of the RNAs, consists of two large subdomains. The 5′ subdomain has been conserved during evolution and appears to be functionally important for the binding of the EF-1 · GTP · aminoacyl-tRNA complex in eukaryotes. The 3′ subdomain has diverged widely between eubacteria and eukaryotes and has produced the 4.5 S RNA in the chloroplast ribosomes of flowering plants.The structure of domain VI within the eubacterial RNAs was probed with chemical reagents in order to establish the degree of stacking and/or accessibility of each adenosine, cytidine and guanosine residue; the double-helical segments were localized with the cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were detected with the single-strand-specific ribonucleases A, T₁ and T₂. The data enabled the three secondary structural models, proposed for the E. coli 23 S RNAs, to be examined critically and it was concluded that many of their structural features are correct. Various differences between the models were considered and evidence is provided for additional structuring in the RNA including the stacking of juxtaposed purines into double helices. The 5′ subdomain constitutes a compact and resistant structure whereas the 3′ subdomain is relatively accessible and contains most of the potential protein binding sites. Moreover, comparison of our results with the published results on 4.5 S RNA suggests that the latter forms essentially the same structure as the 3′ subdomain, in contrast to earlier conclusions.A high level of structural conservation has occurred throughout the RNA domain during the evolution of the Gram negative and Gram positive bacteria although the thermophile was generally more stable at base-pairs adjacent to the terminal loops.     

Ordered water structure around a B-DNA dodecamer: A quantitative study

The crystal structure of the double-helical B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G has been solved and refined independently in three forms: (1) the parent sequence at room temperature; (2) the same sequence at 16 K; and (3) the 9-bromo variant C-G-C-G-A-A-T-T^BrC-G-C-G at 7 °C in 60% (v/v) 2-methyl-2.4-pentanediol. The latter two structures show extensive hydration along the phosphate backbone, a feature that was invisible in the native structure because of high temperature factors (indicating thermal or static disorder) of the backbone atoms. Sixty-five solvent peaks are associated with the phosphate backbone, or an average of three per phosphate group. Nineteen other molecules form a first shell of hydration to base edge N and O atoms within the major groove, and 36 more are found in upper hydration layers. The latter tend to occur in strings or clusters spanning the major groove from one phosphate group to another. A single spermine molecule also spans the major groove. In the minor groove, the zig-zag spine of hydration that we believe to be principally responsible for stabilizing the B form of DNA is found in all three structures. Upper level hydration in the minor groove is relatively sparse, and consists mainly of strings of water molecules extending across the groove, with few contacts to the spine below. Sugar O-1′ atoms are closely associated with water molecules, but these are chiefly molecules in the spine, so the association may reflect the geometry of the minor groove rather than any intrinsic attraction of O-1′ atoms for hydration. The phosphate O-3′ and O-5′ atoms within the backbone chain are least hydrated of all, although no physical or steric impediment seems to exist that would deny access to these oxygen atoms by water molecules.     

A spectrophotometric study of the secondary structure of ribonucleic acid isolated from the smaller and larger ribosomal subparticles of rabbit reticulocytes

The spectrum of RNA from the smaller and larger subparticles of rabbit reticulocyte ribosomes was studied as a function of pH, ionic strength, urea concentration and temperature. It was inferred that both RNA species form short double-helical segments of not more than about 10 base-pairs in length. Not more than about 70% of the base residues may be located in double-helical segments. RNA from the larger subparticle is richer in guanine and cytosine residues and its secondary structure is the more stable. These conclusions are based on the use of double-helical RNA from virus-like particles and of unfractionated Escherichia coli tRNA as model systems.     

Solution conformation of DNA

Optical observations on linear B-form DNA by the method of electric linear dichroism show that the value of the limiting reduced dichroism is molecular weight-dependent, increasing with molecular weight to a limit of about ?1.41 ± 0.02 in aqueous solution. These data and the rotational relaxation times obtained from the decay of the dichroism when the orienting field is instantaneously removed, imply the existence of a non-linear tertiary equilibrium structure for DNA. The data indicate that the essential B-form parameters of the double-stranded DNA are retained in this tertiary structure, and are not consistent with a DNA structure in which the base-pairs have a 34 ° propeller-like twist (Hogan et al., 1978). The interpretation of the dichroism data is supported by a clear demonstration that the magnitude of dichroism change in ethanol corresponds to that expected for the B to A-form structural transition as determined from X-ray diffraction. We propose that the tertiary structure of B-DNA is a helical coil and suggest the limits of the structural parameters of the coil consistent with the observations.     

X-ray diffraction studies of the RNA tetramer GpGpCpUp

The synthetic RNA tetramer GpGpCpUp has been studied by X-ray diffraction of single crystals and fibres. A preliminary crystallographic analysis of single crystals implies that two GpGpCpUp strands form a short antiparallel double helix with G · U base-pairs at the ends of it. As diffraction intensities of single crystals fall into decay, the diffraction pattern gradually changes into a fibre pattern similar to that of A-RNA or of the A form of DNA.     

Structure of the poly(2-thiouridylic) acid duplex

The crystal and molecular structures of poly(2-thiouridylic) acid are remarkably similar to those of A-DNA. How this might be contrived with homopolymer duplexes containing either symmetric or asymmetric pyrimidine pyrimidine base-pairs is described using molecular models optimized by the linked-atom least-squares method. This study supplements an earlier manual model-building study of the second possibility by Mazumdar et al. (1974).     

Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G

The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Conformation and dynamics in a Z-DNA tetramer

Two kinds of conformational variability have been reported for left-handed Z-DNA: the Z to Z′ transition, which involves a change in guanine sugar pucker from C-3′-endo to C-1′-exo, and the Z_Ito Z_II transition, which corresponds to a simple three-atom phosphate-group rotation. Neither of these motions substantially affects base stacking or helical twist, and this is because the degree of independent motion of phosphate groups, sugar molecules and base-pairs is greater in the left-handed Z helix than in right-handed B-DNA. Detailed considerations of Z helix geometry suggest that Z_I, Z_IIand Z′ are not separate species, but only samplings of the full range of conformation open to Z-DNA.     

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.     

Letter to the editor: The polyuridylic acid complex with polyamines: an x-ray fiber diffraction observation.

The poly(U)-spermine complex has been studied by X-ray fiber diffraction techniques. The X-ray pattern is generally similar to that of A RNA or of the A form of DNA, suggesting a double-helical structure with strands of opposite polarity. Models with 9 to 11 residues per turn of the helix are considered.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

MPD and DNA Bending in Crystals and in Solution

Bending of 15 to 24° is observed within crystal structures ofB-DNA duplexes, is strongly sequence-dependent, and exhibits no correlation with the concentration of MPD (2-methyl-2,4-pentanediol) in the crystallizing solution. Two types of bends are observed: facultative bends or flexible hinges at junctions between regions of G·C and A·T base-pairs, and a persistent and almost obligatory bend at the center of the sequence R-G-C-Y. Only A-tracts are characteristically straight and unbent in every crystal structure examined to date. A detailed examination of normal vector plots for individual strands of a double helix provides an explanation, in terms of the stacking properties of guanine and adenine bases. The effect of high MPD concentrations, in both solution and crystal, is to decrease local bending somewhat without removing it altogether. MPD gel retardation experiments provide no basis for choosing among the three models that seek to explain macroscopic curvature of DNA by means of microscopic bending: junction bending, bent A-tracts, or bent general- sequence DNA. Crystallographic data on the straightness of A-tracts, the bendability of non-A sequences, and the identity of inclination angles in A-tract and non-A-tractB-DNA support only the general-sequence bending model. The pre-melting transition observed in A-tract DNA probably represents a relaxation of stiff adenine stacks to a flexible conformation more typical of general-sequence DNA.