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.     

Visualization of drug-nucleic acid interactions at atomic resolution. IV. Structure of an aminoacridine--dinucleoside monophosphate crystalline complex, 9-aminoacridine--5-iodocytidylyl (3'--5') guanosine

9-Aminoacridine forms a crystalline complex with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, space group P2₁ with $\text{a = 13.98}\text{A}\text{?}\text{, b = 30.58}\text{A}\text{?}\text{, c = 22.47}\text{A}\text{?}$ and β = 113.9 °. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by a combination of Fourier and sum-function Fourier methods. The asymmetric unit contains four 9-aminoacridine molecules, four iodoCpG molecules and 21 water molecules, a total of 245 atoms. 9-Aminoacridine demonstrates two different intercalative binding modes and, along with these, two slightly different intercalative geometries in this model system.The first of these is very nearly symmetric, the 9-amino group lying in the narrow groove of the intercalated base-paired nucleotide structure. The second shows grossly asymmetric binding to the dinucleotide, the 9-amino group lying in the wide groove of the structure. Associated with these two different intercalative binding modes is a difference in geometries in the structures. Although both structures demonstrate C3′ endo (3′–5′) C2′ endo mixed sugar puckering patterns (i.e. both cytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), with corresponding twist angles between base-pairs of about 10 °, they differ in the magnitude of the helical screw axis dislocation accompanying intercalation (Sobell et al., 1977a,b). In the pseudosymmetric intercalative structure, this value is about +0.5 Å, whereas in the asymmetric intercalative structure this value is about +2.7 Å. These conformational differences can be best described as a “sliding” of base-pairs on the intercalated acridine molecule.Although the pseudosymmetric intercalative structure can be used in 9-aminoacridine-DNA binding, the asymmetric intercalative structure cannot since this poses stereochemical difficulties in connecting neighboring sugar-phosphate chains to the intercalated dinucleotide. It is possible, however, that the asymmetric binding mode is related to the mechanism of 9-aminoacridine-induced frameshift mutagenesis (Sakore et al., 1977), and we discuss this possibility here in further detail.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Solvent-accessible surfaces of nucleic acids

Static solvent-accessible surface areas were calculated for DNA and RNA double helices of varied conformation, composition and sequence, for the single helix of poly(rC), and for a transfer RNA. The results show that for DNA and RNA double helices, two thirds of the water-accessible surface area become buried on double helix formation; phosphate oxygens retain near maximal exposure while the bases are 80% buried. Transfer RNA exposes slightly less surface per residue than does double-helical RNA, despite the presence of several additional “modified” groups, all of which are exposed significantly.When a probe corresponding to a single water molecule is used, both the total and atom type exposures are very similar for A-DNA and B-DNA, although marked differences appear in the major and minor groove exposures between the two conformations. For a given base-pair, the accessible surface area buried upon double-helical stacking is nearly constant (within 5%) for different sequences of neighboring base-pairs.For probes larger than single water molecules, there exist considerable differences in the total and atom type exposures of A-DNA and B-DNA. Conformational transitions between the A-DNA and B-DNA helical forms can thus be related to differences in the accessible areas for “structured” water, or a secondary hydration shell, rather than to interactions with individual water molecules of the primary hydration shell. The base-composition dependence of DNA helical conformation can be explained in terms of the opposing effects of thymine methyl groups of A · T base-pairs and the amino groups of G · C base-pairs upon the solvent within the grooves.The area calculations show that primarily the major groove of B-DNA and the minor groove of A-DNA have sufficient accessible surface area to be recognized by a probe size corresponding to the side-chains of amino acids.     

Visualization of drug-nucleic acid interactions at atomic resolution: II. Structure of an ethidium/dinucleoside monophosphate crystalline complex,ethidium:5-iodocytidylyl (3′–5′) guanosine

Ethidium forms a second crystalline complex with the dinucleoside monophosphate 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, P2₁, with $\text{a = 14.06}\text{A}\text{?}\text{, b = 32.34}\text{A}\text{?}\text{, c = 16.53}\text{A}\text{?}\text{, β = 117.8 °}$. The structure has been solved to atomic resolution using rigid-body Patterson vector search and Fourier methods, and refined by full matrix least-squares to a residual of 0.16 on 3180 observed reflections. The structure consists of two ethidium molecules, two iodoCpG molecules, 27 water molecules and four methanol molecules, a total of 165 atoms (excluding hydrogens) in the asymmetric unit. Both iodoCpG molecules are hydrogen-bonded together by guanine · cytosine Watson-Crick base-pairing. Adjacent base-pairs within this paired iodoCpG structure and between neighboring iodoCpG molecules in adjoining unit cells are separated by 6.7 Å. This distance reflects the presence of an ethidium molecule intercalated between base-paired iodoCpG molecules and another ethidium molecule stacked above (and below) the dinucleotide. Approximate 2-fold symmetry is used in the interaction; this reflects the pseudo-2-fold symmetry axis of the phenanthridinium ring system in ethidium coinciding with the approximate 2-fold axis relating base-paired iodoCpG molecules. The phenyl and ethyl groups of the intercalated ethidium molecule lie in the narrow groove of the miniature iodoCpG double-helix. The stacked ethidium, however, lies in the opposite direction, its phenyl and ethyl groups neighboring iodine atoms on cytosine residues. Base-pairs within the paired nucleotide units are related by a twist of about 8 °. The magnitude of this angular twist reflects conformational changes in the sugar-phosphate chains accompanying intercalation. These primarily reflect the differences in ribose sugar ring puckering that are observed (i.e. both iodocytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), and alterations in the glycosidic torsional angles that describe the base-sugar orientation.The information provided by this structure analysis (along with the accompanying one (ethidium:iodoUpA), described in the previous paper) has led to an understanding of the general nature of intercalative drug binding to DNA. This is described in the third paper of this series.     

Ligation of highly modified bacteriophage DNA

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4′,5′-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, λ DNA to be ligated was as follows: $\text{λ}\text{DNA}\text{=}\text{XP}\text{12}\text{DNA}\text{>}\text{SP}\text{82}\text{DNA}\text{?}\text{nonglucosylated}\text{T}\text{4}\text{DNA}\text{>}\text{T}\text{4}\text{DNA}\text{=}\text{PBS}\text{1}\text{DNA}\text{?}\text{SP}\text{15}\text{DNA}$. TaqI-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and λ DNAs was equally efficient. We conclude that the poor ligation of TaqI fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.     

Visualization of drug-nucleic acid interactions at atomic resolution: I. Structure of an ethidium/dinucleoside monophosphate crystalline complex,ethidium:5-Iodouridylyl (3′–5′) adenosine

Ethidium forms a crystalline complex with the dinucleoside monophosphate 5-iodouridylyl(3′–5′)adenosine (iodoUpA). These crystals are monoclinic, space group C2, with unit cell dimensions, $\text{a = 28.45}\text{A}\text{?}\text{, b = 13.54}\text{A}\text{?}\text{, c = 34.13}\text{A}\text{?}\text{, β = 98.6 °}$. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares to a residual of 0.20 on 2017 observed reflections. The asymmetric unit contains two ethidium molecules, two iodoUpA molecules and 27 water molecules, a total of 155 atoms excluding hydrogens. The two iodoUpA molecules are held together by adenine · uracil Watson-Crick-type base-pairing. Adjacent base-pairs within this paired iodoUpA structure and between neighboring iodoUpA molecules in adjoining unit cells are separated by about 6.7 Å; this separation results from intercalative binding by one ethidium molecule and stacking by the other ethidium molecule above and below the base-pairs. Non-crystallographic 2-fold symmetry is utilized in this model drug-nucleic acid interaction, the intercalated ethidium molecule being oriented such that its phenyl and ethyl groups lie in the narrow groove of the miniature nucleic acid double-helix. Base-pairs within the paired nucleotide units are related by a twist of 8 °. The magnitude of this angular twist is related to conformational changes in the sugar-phosphate chains that accompany drug intercalation. These changes partly reflect the differences in ribose sugar ring puckering that are observed (both iodouridine residues have C3′ endo sugar conformations, whereas both adenosine residues have C2′ endo sugar conformations), and alterations in the glycosidic torsional angles describing the base-sugar orientations. Additional small but systematic changes occur in torsional angles that involve the phosphodiester linkages and the C4′C5′ bond. Solution studies have indicated a marked sequence-specific binding preference in ethidium-dinucleotide interactions, and a probable structural explanation for this is provided by this study.This structure and the accompanying one described in the second paper [ethidium:5-idocytidylyl(3′–5′)guanosine] are examples of model drug-nucleic acid intercalative complexes, and the information provided by their structure analyses has led to a general understanding of intercalative drug binding to DNA. This is described in the third paper of this series.     

Crystal structure of an inhibitor and a model for inhibition of replicative DNA synthesis

6-(p-Hydroxyphenylhydrazino)-uracil is an antimicrobial agent that selectively blocks replicative DNA synthesis in Bacillus subtilis by inhibiting DNA polymerase III. The drug crystallizes as a monoclinic monohydrate, space group $\text{C2}\text{c}$, with $\text{a = 23.920(6)}\text{A}\text{̊}\text{, b = 5.587(3)}\text{A}\text{̊}\text{, c = 17.466(5)}\text{A}\text{̊}\text{, β = 101.45(8) °}$, and eight hydrated molecules per cell. Three-dimensional X-ray diffraction data were collected. The structure was solved by Patterson methods and refined to an R value of 6.8% for the 1651 data. The geometry of the uracil ring is unusual. The bond distances suggest that a resonance form involving a positively charged hydrazino nitrogen and a negatively charged carbonyl oxygen, O(4), makes a large contribution to the valence bond structure of this compound. The exocyclic C(6)N bond is short (1.335 Å), the C(6)C(5) bond distance is 1.371 Å, which is longer than in uracil, and the C(5)C(4) distance (1.396 Å) is short. The uracil ring, the linked hydrazino nitrogen, and the hydrogen on this nitrogen are in the same plane. Each uracil group is hydrogen bonded to a nearly coplanar uracil across a center of symmetry. The water molecule is also near the plane of these paired bases and forms a hydrogen bond with the uracil-linked hydrazino NH group. This paired base arrangement and the restricted rotation about the exocyclic C(6)N link that constrains the hydrazino NH group to lie near the uracil plane suggest a model for the interaction of the drug with template-primer DNA. The drug acts when cytosine is the base to be copied in the template strand, and the drug is competitive with dGTP. Both cytosine and guanine can be accommodated with little distortion of the crystal structure geometry in a manner compatible with the known geometry of DNA. The structural and biochemical aspects of the model for drug action are discussed.     

Methoxyspheroidene and methoxyspheroidenone,two carotenoids from Rhodopseudomonas spheroides

Two new carotenoids isolated from Rhodopseudomonas spheroides (Rhodospirillaceae) have been identified as methoxyspheroidene (1,1′-dimethoxy-3,4-didehydro-1,2,1′,2′,7$\text{,}\text{?}$8′-hexahydro-ψ,ψ-carotene) obtained from anaerobic cultures and methoxyspheroidenone (1,1′-dimethoxy-3,4-didehydro-1,2,1′,2′,7′,8′-hexahydro-ψ,ψ-caroten-2-one) recovered from aerobic cultures.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Flavonoids of Gardenia cramerii and G. fosbergii bud exudates

From the bud exudates of Gardenia cramerii and G. fosbergii, two species endemic to Sri Lanka, a new flavonoid with an unusual B-ring oxidation pattern, 5,5′-dihydroxy-6,7,2′,3′-tetramethoxyflavone, was characterized. Two other rare flavonoids, 5,3′,5′-trihydroxy-3,6,7,4′-tetramethoxyflavone and 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone were also isolated from both Gardenia species.     

Preliminary crystaliographic data for glyceraldehyde-3-phosphate dehydrogenase from the thermophile Bacillus coagulons

Crystals of thermolabile glyceraldehyde-3-phosphate dehydrogenase from Bacillus coagulans suitable for high resolution X-ray crystallographic analysis have been grown from sodium citrate solutions by equilibrium dialysis. The space group is C222₁, with cell dimensions $\text{a = 95·6 (2)}\text{A}\text{?}\text{, b = 137·2 (3)}\text{A}\text{?}\text{and}\text{c = 131·9 (4)}\text{A}\text{?}$. The molecules have one crystallographically exact 2-fold rotation axis of symmetry.     

A new solid-state microelectrode for measuring intra-cellular chloride activities

Solid-state microelectrodes for measuring intracellular Cl^? activity (aⁱCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 μm), coating them under vacuum with a 0.2–0.3 μm thick layer of spectroscopic grade silver, and sealing them (except for the terminal 2–5 μm of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0 ± 0.6 mV (S.E.) per decade change in α_Cl. Tip resistance was (77.1 ± 3.1 × 10⁹ω (n=30). Electrode response was rapid (10–20 s), was unaffected by HCO₃^?, H₂PO₄^2? or protein, and remained essentially unchanged over a 24-h period. αⁱ_Cl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, αⁱ_Cl significantly exceeded the value corresponding to equilibrium distribution of Cl^? across the cell membrane.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

The enzymatic sulphation of heparan sulphate by hen's uterus

A sulphotransferase preparation from hen's uterus catalysed the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to N-desulphated heparan sulphate, heparan sulphate, N-desulphated heparin and dermatan sulphate. Heparin, chondroitin sulphate and hyaluronic acid were inactive as substrates for the enzyme. N-desulphated heparin was a much poorer substrate for the enzyme than N-desulphated heparan sulphate suggesting that properties of the substrate other than available glucosaminyl residues influenced enzyme activity. N-acetylation of N-desulphated heparin and N-desulphated heparan sulphate reduced their sulphate acceptor properties so it was unlikely that the N-acetyl groups of heparan sulphate facilitated its sulphatiion. Direct evidence for the transfer of [³⁵S]sulphate to amino groups of N-desulphated haparan sulphate was obtained by subsequent isolation of glucosamine $\text{N-[}{}^{35}\text{S}\text{]}\text{sulphate}$ from heparan [³⁵S]sulphate product. This was made possible through the use of a flavobacterial enzyme preparation which contained “heparitinase” activity but had been essentially freed of sulphatases. Attempts to transfer [³⁵S]sulphate to glucosamine or N-acetylglucosamine were unsuccessfull.     

A tetragonal crystal form of horse spleen apoferritin and its relationship to the cubic modification

Horse spleen apoferritin has been crystallized as tetragonal plates and needles with a unit cell with $\text{a = b = 147 ± 0.5}\text{A}\text{?}\text{and}\text{c = 154.4 ± 0.5}\text{A}\text{?}$. The space group is P42₁2 and the unit cell contains two molecules in a pseudo-body-centred arrangement. The intensity distributions and calculated rotation functions of tetragonal and cubic crystals have been compared. The symmetry of the diffraction patterns from cubic crystals indicates that the molecules have 432 symmetry with their 4-fold axes lying along the cube axes. In the tetragonal crystals one molecular 4-fold axis lies parallel to c, the unique axis, while the rest of the molecular point symmetry is not used by the lattice. Instead the remaining 4-fold axes of the two molecules, which lie in planes perpendicular to c, are rotated ± 17.5 ° with respect to the tetragonal a axis. The finding that apoferritin reassembled from subunits can be crystallized in both tetragonal and cubic forms confirms its conformational similarity to native molecules.     

The influence of adenosine 3′,5′-monophosphate upon the activity of the membrane-associated (Ca2++Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

The phosphohydrolase activity of the membrane-associated $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+Mg}{}^{\mathrm{2+}}\text{)-dependent}$ adenosine triphosphatase (ATPase) of the human erythrocyte can be inhibited by micromolar or nanomolar concentrations of cyclic AMP. Millimolar concentrations of cyclic AMP are less effective. The inhibitory effect of cyclic AMP is potentiated in the presence of the phosphodiesterase inhibitor, theophylline.