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The radicals induced by X-irradiation at 300 K of single crystals of uracil have been analysed by E.S.R. spectroscopy. Immediately after irradiation, three radicals are detected: one hydrogen abstraction and two hydrogen addition radicals. The hydrogen abstraction is from N1 and the hydrogen addition occurs at position C5 and at position O4. Upon subsequent illumination with light of wavelength λ > 400 nm, the C5-addition radical transforms into C6-addition where the added hydrogen atom is localized on carbon C6. As in ohter pyrimidines, the β-proton splitting of the C5-addition radical is much smaller than that of the C6-addition radical (35·5 G instead of 48·0 G).

With the use of PMR the ribose conform ations have been studied in the tem perature range - 60 to + 40 °C in ND3 solutions of adenosine (A), guanosine (G), inosine (I), xanthosine (X), purineriboside (P R), 2-am inopurineriboside (2am P R), N6-isopentenyladenosine (N6ipA), 8-bromo-adenosine (8-BrA), 8-bromoguanosine (8-BrG), formycin B (F), tubercidin (T), isopropylidene-adenosine (iA), and isopropylideneguanosine (iG). The analysis is based on the two-state S ⇄ N model of the ribose m oiety proposed by Altona and Sundaralingam . The compounds studied can be classified into two groups: 1. A, I, G, X, PR , 2am PR, N6ipA, and T show a small tem perature dependence of the S ⇄ N equilibrium and [ S ] ~ 0 .6 ; 2. 8-BrA, 8-BrG, and F have a stronger tem perature dependence and [S] ~ 0 .8 . W ithin these two groups the sim ilarities observed are greater than observed in the solid state. Some therm odynam ic conclusions about the S ⇄ N and the syn ⇄ anti equilibria are presented. The results support the previously proposed correlation of the S state of the ribose with the syn conform ation of the base and of the N state of the ribose with the anti conform ation of the base. Furtherm ore, it is derived that the gg rotam er is correlated with the S state of the ribose and therefore stabilizes the syn conformation of the base.

PERSPECTIVES AND OVERVIEW 125 SOLVENT LOCATION BY CRYSTALLOGRAPHIC REFINEMENT 126 Structural Water in Refinement 127 Water-Mediated Bridges and Crystal Stability 129 HYDRATION OF NUCLEIC ACID HELICES... ....... .•••••••.....•.•• .•.• • • 130 Structural Hydration Motifs .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Internal Water in Transfer RNAs .... 133 Water and Dielectric Screening 134 Water Activity and DNA Conformation 136

Radicals produced in x‐irradiated single crystals of L‐histidine free base in the temperature range 77–300 K have been investigated using ESR spectroscopy at 9.5 and 35 GHz. The main species trapped at 77 K are radical anions located at the carboxyl group. These are converted to deamination radicals upon warming. Conversion of the anion into the deamination radical is not found if the former is bleached by uv illumination at 77 K. At room temperature, only radicals resulting from deamination are observed. Two different conformations of the deamination have been found. The one obtained at room temperature is produced from a precursor with identical composition but different conformation. The latter, which is stable at reduced temperatures, is in the open conformation like both the precursor anion radical and the parent molecule. The room temperature radical however adopts a closed conformation. The identifications are supported by MO‐SCF calculations at the INDO level. A detailed analysis of the anion radical and the deamination radical by INDO is presented.

The results of MO INDO calculations of some thymine radicals are presented. These include as well as the hydrogen addition radicals the hydroxyl addition radicals to C5 and to C6 of the C5=C6 double bond of the thymine molecule. The radicals resulting from hydrogen abstraction from the methyl group and from the nitrogen N1 are also studied. Except for radicals where the main spin density is localized on nitrogen atoms, the agreement between experimental and theoretical couplings is satisfactory.

The quantity of data generated from molecular dynamics simulations and energy minimizations of macromolecules is overwhelming. It is an arduous task to extract the relevant and interesting information from the numerous coordinate sets produced. To help solve this problem, the authors have developed a method to aid the visualization of the relevant information from the simulations. This approach combines animation of the results on a high performance graphics device, such as the PS300, with colour-coded atoms based on changes in energy or conformation. The method will be illustrated using as examples: the molecular mechanics minimization of a nonapeptide, the molecular dynamics simulation of the protein myoglobin, including the analysis of the motion of helices during a 300ps trajectory, and changes in sugar puckering that occur during the molecular dynamics simulation of a DNA oligomer. The method is also applicable for analysing energy components and conformational properties of a fixed conformation.

Four crystal structures of transfer RNA molecules were refined at 3 A resolution with the inclusion of the solvent molecules found in the difference maps: yeast tRNA-phe in the orthorhombic form, yeast tRNA-phe in the monoclinic form and yeast tRNA-asp in the A and B forms. Over 100 solvent molecules were located in each tRNA crystal. Several hydration schemes are found repeatedly in the 4 crystals. The tertiary interactions in the corner of the L-shaped molecule attract numerous solvent molecules which bridge the ribose hydroxyl O(2') atoms, base exocyclic atoms and phosphate anionic oxygen atoms. Conservation of bases leads to conservative localized hydration patterns. Several solvent molecules are found stabilizing unusual base pairs like the G-U pairs and those involving the pseudouridine base. Water bridges between the O(2') and the exocyclic atom O2 of pyrimidines or the N3 atom of purines are common. Water bridges occur frequently between successive anionic oxygen atoms of each strand as well as between N7 or other exocyclic atoms of successive bases in the major groove. Magnesium ions or spermine molecules are found to bind in the major groove of tRNA helices without specific interactions.

The solvent molecules found around crystallized oligonucleotides after X-ray refinement are analysed in terms of interaction sites to bases, phosphates and sugars in the three main forms of nucleic acid structures, the A-form, the B-form and the Z-form. The average numbers of contacts to nucleic acid atoms made by solvent molecules are identical in the three forms, but it appears that the average number of contacts solvent molecules make with each other depends on the resolution of the structure. The phosphate anionic oxygen atoms are the most hydrated, while the O(3′) and O(5′) backbone atoms and the ring oxygen atom O(4′) are the least hydrated. Among the hydrophilic atoms of the bases, there is a modulation of the relative water affinities with the nucleic acid form. Numerous hydration sites are such that water molecules can bridge hydrophilic atoms of the same residue, of adjacent residues on the same strand, of distant residues on the two strands, or belonging to symmetry-related residues. Through the helical periodicity of the nucleic acid structure, those bridges can lead to regular and striking hydration networks involving several water molecules and characteristic of the nucleic acid form. Solvent dynamics, as seen by temperature factor versus occupancy plots, seems intimately related to nucleic acid structure and dynamics, since they depend on hydration sites around the nucleic acids.

Four transfer RNA crystals, the monoclinic and orthorhombic forms of yeast tRNA(Phe) as well as forms A and B of yeast tRNA(Asp), have been submitted to the same restrained least-squares refinement program and refined to an R factor well below 20% for about 4500 reflections between 10 and 3 A. In yeast tRNA(Asp) crystals the molecules exist as dimers with base pairings of the anticodon (AC) triplets and labilization of the tertiary interaction between one invariant guanine of the dihydrouridine (D) loop and the invariant cytosine of the thymine (T) loop (G19-C56). In yeast tRNA(Phe) crystals, the molecules exist as monomers with only weak intermolecular packing contacts between symmetry-related molecules. Despite this, the tertiary folds of the L-shaped tRNA structures are identical when allowance is made for base sequence changes between tRNA(Phe) and tRNA(Asp). However, the relative mobilities of two regions are inverse in the two structures with the AC loop more mobile than the D loop in tRNA(Phe) and the D loop more mobile than the AC loop in tRNA(Asp). In addition, the T loop becomes mobile in tRNA(Asp). The present refinements were performed to exclude packing effects or refinement bias as possible sources of such differential dynamic behavior. It is concluded that the transfer of flexibility from the anticodon to the D- and T-loop region in tRNA(Asp) is not a crystal-line artefact. Further, analysis of the four structures supports a mechanism for the flexibility transfer through base stacking in the AC loop and concomitant variations in twist angles between base pairs of the anticodon helix which propagate up to the D- and T-loop region.

Radical formation by irradiation with X-rays in single crystals of 1-methyluracil has been measured using ESR-spectroscopy at 9.5 GHz and 35 GHz. When irradiating and measuring at 77 °K radicals characterized by hydrogen abstraction from the methyl group were found to be predominating. For the hyperfine splittings principal values of 8.5 G, 18.2 G, 30.5 G were found for both α-protons and 3.3 G, 2.4 G, 1.7 G for the N (1)-splitting. Pairs of abstraction radicals were also found under these conditions showing quintet splittings of about 10 G and anisotropic dipolar coupling. Average distances parallel to c of 6.3 Å and 6.5 Å were found for two types of pairs while those perpendicular to c were not analyzed in detail. Irradiating and measuring at room temperature an additional sextet pattern was found in the spectrum and attributed to a radical formed by addition of hydrogen at C (5). For the hyperfine splittings principal values of 8.1 G, 17.5 G, 30.0 G were found for the α-proton and 31.2 G, 34.4 G, 34.1 G for the two equivalent β-protons. Radical concentrations were determined at 77 °K yielding about 90% abstraction radical, 15% of these in pairs and 10% of addition radical. About half of the abstraction radicals are converted to addition radicals between 200 °K and 280 °K. The latter disappear at about 460 °K leaving a fraction of abstraction radicals which is annealed at even higher temperatures.

SummaryRadical formation in monocrystals of 9-ethyl adenine by irradiation with X-rays at room temperature has been studied using e.s.r.-spectroscopy at 9·5 GHz and 35 GHz. The two types of spectra found were attributed to hydrogen abstraction and addition radicals. It was possible to separate the correlated spectra and to assign definite structures to the two species by analysing the orientational variations of the spectra. The spectra of the abstraction radical were obtained under conditions of power saturation of the addition radical at reduced temperature (77 K). The site of hydrogen dissociation and of maximum unpaired spin density was localized at the C-10-carbon atom bound to N-9 of the purine. The methyl β-protons give isotropic splittings of 24·0 G each. The abstraction radical could be bleached by illumination with U.V.-light, leaving the addition radical unchanged. Hydrogen addition was concluded on the basis of isotropic hyperfine interactions from two β-protons of 39·5 G each and anisotropic ...

crystals of cytosine. The resuits were compatible with the presence of H-addition radicals. One was 5,6-dihydro-cytosine6yl resulting from H-addition at C-5; it is suggested that the other could result from H-addition to O-7. The work is briefly discussed in relation to spectral differences found by other workers. (UK)

SummaryLow-temperature irradiation and warming-up experiments were used to study by e.s.r. the radical transformation in irradiated cytosine and its derivatives. It is shown that the H adduct radical of the base observed at room temperature is the result of the protonation of the base-anion radical observed at low temperature. In the nucleoside and nucleotide, this protonation occurs around 230 K. The main sources of protons are the molecules of crystallization and the sugar group. The different mechanisms proposed for the radiolysis of DNA constituents are discussed in connection with these findings.

SummaryRadical formation in monocrystals of equimolar complexes of 9-ethyl adenine: 1-methyl uracil by irradiation with X-rays was investigated at 77 K and 300 K using e.s.r.-spectroscopy at 9·5 GHz and 35 GHz. Five different types of spectrum were identified, which corresponded to spectra of the irradiated components. A hydrogen abstraction radical (I and III) and a hydrogen addition radical (II and IV) were assigned to each of the two constituents. The fifth species, with an unidentified singlet after irradiation at 77 K, has also been found in irradiated single crystals of 9-ethyl adenine. By bleaching with U.V.-light or heating to about 150 K, it is decomposed irreversibly. After a crystal irradiated at 77 K is warmed to room temperature only pyrimidine radicals I and II are left. At 77 K the two types of radical (I and II) in 1-methyl uracil are present in addition to the singlet line; at 300 K the addition radicals (II and IV) predominate in comparable proportions, but the abstraction radicals (I an...

Aminoglycoside antibiotics inhibit protein biosynthesis and various ribozymes. Structural electrostatic complementarity can explain the inhibition mechanism of the hammerhead ribozyme: positively charged ammonium groups match the negatively charged metalion-binding pockets created by the RNA fold's electrostatic field.

The natural bases of nucleic acids form a great variety of base pairs with at least two hydrogen bonds between them. They are classified in twelve main families, with the Watson–Crick family being one of them. In a given family, some of the base pairs are isosteric between them, meaning that the positions and the distances between the C1′ carbon atoms are very similar. The isostericity of Watson–Crick pairs between the complementary bases forms the basis of RNA helices and of the resulting RNA secondary structure. Several defined suites of non-Watson–Crick base pairs assemble into RNA modules that form recurrent, rather regular, building blocks of the tertiary architecture of folded RNAs. RNA modules are intrinsic to RNA architecture are therefore disconnected from a biological function specifically attached to a RNA sequence. RNA modules occur in all kingdoms of life and in structured RNAs with diverse functions. Because of chemical and geometrical constraints, isostericity between non-Watson–Crick pairs is restricted and this leads to higher sequence conservation in RNA modules with, consequently, greater difficulties in extracting 3D information from sequence analysis. Nucleic acid helices have to be recognised in several biological processes like replication or translational decoding. In polymerases and the ribosomal decoding site, the recognition occurs on the minor groove sides of the helical fragments. With the use of alternative conformations, protonated or tautomeric forms of the bases, some base pairs with Watson–Crick-like geometries can form and be stabilized. Several of these pairs with Watson–Crick-like geometries extend the concept of isostericity beyond the number of isosteric pairs formed between complementary bases. These observations set therefore limits and constraints to geometric selection in molecular recognition of complementary Watson–Crick pairs for fidelity in replication and translation processes.

Molecular dynamics simulations have been used to investigate the binding of Mg 2+ ions to the deep groove of the eubacterial 5S rRNA loop E. The simulations suggest that long-lived and specific water-mediated interactions established between the hydrated ions and the RNA atoms lining up the binding sites contribute to the stabilization of this motif. The Mg 2+ binding specificity is modulated by two factors: (i) a required electrostatic complementarity and (ii) a structural correspondence between the hydrated ion and its binding pocket that can be estimated by its degree of dehydration and the resulting number and lifetime of the intervening water-mediated contacts. Two distinct binding modes for pentahydrated Mg 2+ ions that result in a significant freezing of the tumbling motions of the ions are described, and mechanistic details related to the stabilization of nucleic acids by divalent ions are provided.

The INDO method has been used to calculate the 13 C and β-proton coupling constants of some carboxyl anion radicals. It is concluded that the deviation from planarity in these radicals is approximately 6°. It is proposed that these radicals constitute models for the formation of tetrahedral intermediates in addition reactions of a nucleophile to a carbonyl group.

Two recent reports [1, 2] describe the stunning crystal structures of complexes between a viral protein that suppresses RNA silencing and a 21 nucleotide small interfering (si)RNA.

On the morning of December 26, 2004, Muttaiya Sundaralingam and his wife Indrani did not survive the major tsunami as it hit the coast of Sri Lanka in Trincomalee following the submarine earthquake which had occurred a couple of hours earlier off the coast of Sumatra. Dr. Sundaralingam left us with central contributions in the field of the stereochemistry of nucleic acids. He was also a major player in the development of the crystallography of nucleic acids from single bases to large macromolecules.

SummaryThe radicals produced by X-irradiation in L-phenylalanine ·HCl crystals have been analysed by electron-spin-resonance (e.s.r.) spectroscopy. Four radicals have been identified: the radical resulting from electron capture by the carboxylic group, the radical resulting from deamination, an hydrogen addition radical to the benzene ring, and the radical resulting from hydrogen abstraction from the methylene group bonded to Cβ. These identifications are supported by INDO calculations.

The non-self-complementary dinucleoside monophosphate cytidylyl-3',5'-adenosine (CpA) forms a base-paired parallel-chain dimer with an intercalated proflavine. The dimer complex possesses a right-handed helical twist. The dimer helix has an irregular girth with a neutral adenine-adenine (A-A) pair, hydrogen-bonded through the N6 and N7 sites (C1'...C1' separation of 10.97 A), and a triply hydrogen-bonded protonated cytosine-cytosine (C-C) pair with a proton shared between the base N3 sites (Cl'...Cl' separation of 9.59 A). The torsion angles of the sugar-phosphate backbone are within their most preferred ranges and the sugar puckering sequence (5' leads to 3') is C3'-endo, C2'-endo. There is also a second proflavine molecule sandwiched between CpA dimers on the 21-axis. Both proflavines are necessarily disordered, being on dyad axis, and this suggests possible insights into the dynamics of intercalation of planar drugs. This structure shows that intercalation of planar drugs in nucleic acids may not be restricted to antiparallel complementary Watson-Crick pairing regions and provides additional mechanisms for acridine mutagenesis.

A comparative study of the solution conformation of yeast tRNAAsp and tRNAPhe was undertaken with chemical reagents as structural probes. The accessibility of phosphates was assayed with ethylnitrosourea, that of N7 positions in guanines and adenines with dimethylsulfate and diethylpyrocarbonate and that of the N3 position in cytosines with dimethylsulfate. Statistical modifications were done on end-labelled tRNAs subsequently split at the modified positions and analyzed using sequencing gel methodologies. The results were correlated with the crystal conformations of both tRNAs which were analyzed with a graphic modelling program. Three particular positions are xiscussed. In all tRNAs so far studied, including tRNAAsp and tRNAPhe, phosphate 60 is protected against alkylation, even in the isolated 3’half-molecule of tRNAAsp. This indicates a common T-loop conformation in tRNA which is correlated with the constant presence of residue C61. A similar conformation is found in turnip yellow mosaic virus tRNA-like structure. Adenosine 21 and guanosine 45, two common residues in tRNAAsp and tRNAPhe, exhibit different reactivities against diethylpyrocarbonate and dimethylsulfate. A21 is reactive in tRNAAsp and protected in tRNAPhe; for G45 the situation is inversed. As to A21, the different reactivity is correlated with the extra-loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46.

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.

The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.

The effects of X-ray refinement algorithm on parameters characterising nucleic acid structure are analysed following the re-refinement of the B-dodecamer d(CGCGAATTCGCG). The main conclusions are the following. Mean deviations of main chain torsion angles between the two refinements average 12.6 degrees. Phase angle of pseudorotation for sugar puckers vary between 100 degrees and 180 degrees in the present refinement with amplitude of pucker around 30 degrees. On the other hand, the helical parameters have mean deviations less than 2 degrees. At most half of the assigned solvent positions are within 2 A in both refinements. In the second part of the work, plots of temperature factors (B's) versus occupancies (Q's) for solvent peaks have been analysed in the B-dodecamer and in the Z-hexamer d(5BrCG5BrCG5BrCG). Owing to the poor statistics, some of those conclusions should be regarded as tentative. Occupancy appears to depend on the number of contacts made by the solvent peak with the nucleic acid while temperature factor does not. Except when engaged in particular interaction sites, solvent molecules bound to phosphates have a tendency for high B's and variable Q's. Water molecules bound to polar atoms of the bases occupy various positions in the B-Q diagram. Particularly striking is the behavior of the water molecules belonging to the B-spine and to the Z-spine: the spread in occupancy of water molecules in the hydration spine of the Z-oligomer is larger than in the hydration spine of the B-oligomer. An opposite tendency is observed for the temperature factors. The first observation might reflect the special mobility of the water molecules building up the spine hydration in the Z-form where it continues without interruption from one hexamer to the next. In the B-form, on the contrary, the spine is restricted to the center part of the dodecamer. The second observation might reflect the sharpness of the local attractive potential in the Z-form and its broadness in the B-form. In both cases, dipole reorientations would occur, leading to a high local dielectric constant: in the Z-form, through water molecules hopping from one site to another and, in the B-form, because of rotational freedom.

The interaction of proflavine hemisulfate with the sodium salt of poly(rC-rA) in solution (unbuffered) yields an inverted (mirror-like) circular dichroism (CD) spectrum to that of the free poly(rC-rA). Simultaneously, an induced negative Cotton effect appears in the proflavine band region with a maximum at 467 nm and a slight shoulder at 420 nm. This observation may be explained as resulting from the formation of a poly(rC-rA).proflavine complex with the polynucleotide existing as a right-handed parallel chain duplex with the proflavine intercalated between the CpA sequence and not the ApC sequence. The intercalation geometry here is expected to be analogous to that found in the crystal structure of the dinucleotide CpA.proflavine complex (Westhof et al. J. Mol. Biol., 1981) which forms a miniature right-handed helix. Although normally an inverted spectra could be attributed to a reversal in the helix handedness, the similarity in the 31P nuclear magnetic resonance spectra between the free and proflavine bound poly(rC-rA) indicates that their handedness is the same. The inverted CD spectrum may be a result of the different stacking orientation between the intercalated proflavine and the A-A base-pair on one hand and the triply hydrogen bonded protonated C-C base-pair on the other.

The molecular structure of yeast tRNAAsp, a short extra-loop tRNA, was solved at 3 A resolution in two closely related crystal forms. The resulting model confirms the folding originally found in tRNAPhe: major differences concern the conformations of the loops and the relative positioning of the acceptor and anticodon stems which are more open, conferring to the tRNAAsp molecule its boomerang like shape. Crystal packing involves self-complementary GUC anticodon interactions thus making the structure a tempting model of a tRNA interacting with mRNA on the ribosome. The chemical stability of the tRNA in the crystalline state and in solution is compared using end-labelling and rapid sequencing gel methodologies. Partial splitting of the ribose-phosphate backbone in the anticodon loop was observed in solution but not in the crystal where the splitting is more pronounced in the D-loop.

Detailed atomic models of a eubacterial 5 S rRNA (spinach chloroplast 5 S rRNA) and of a eukaryotic 5 S rRNA (somatic and oocyte 5 S rRNA from Xenopus laevis) were built using computer graphic. Both models integrate stereochemical constraints and experimental data on the accessibility of bases and phosphates towards several structure-specific probes. The base sequence was first inserted on to three-dimensional structural fragments picked up in a specially devised databank. The fragments were modified and assembled interactively on an Evans & Sutherland PS330. Modeling was finalized by stereochemical and energy refinement. In spite of some uncertainty in the relative spatial orientation of the substructures, the broad features of the models can be generalized and several conclusions can be reached: (1) both models adopt a distorted Y-shape structure, with helices B and D not far from colinearity; (2) no tertiary interactions exist between loop c and region d or loop e; (3) the internal loops, in particular region d, contain several non-canonical base-pairs of A.A, U.U and A.G types; (4) invariant residues appear to be more important for protein or RNA binding than for maintaining the tertiary structure. The models are corroborated by footprinting experiments with ribosomal proteins and by the analysis of various mutants. Such models help to clarify the structure-function relationship of 5 S rRNA and are useful for designing site-directed mutagenesis experiments.

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P42212 with cell constants a = b = 19.38(1) A and c = 27.10(1) A . The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities. The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) A) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) A). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche−-gauche−) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche+ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 42-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids. The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.

The temperature-jump method was used to compare the stability of anticodon-anticodon duplexes formed by the self-association of two tRNAs: yeast tRNA-Asp and Escherichia coli tRNA-Gly. Yeast tRNA-Asp duplexes contain a U/U mismatch while E. coli tRNA-Gly dimers have a C/C mismatch in the middle position of their quasi self-complementary anticodons GUC and GCC, respectively. At neutral pH, it is found that only tRNA-Asp duplexes exist whereas at pH 5.0 only tRNA-Gly duplexes are formed. This reflects the hemi- protonation of the N3 of the cytosines at pH 5.0 which induces a pairing between the two middle residues of the anticodon GCC in E. coli tRNA-Gly. This is the first evidence that a protonated C-C(+) base pair is compatible with the formation of a double helix with antiparallel strands in a natural RNA molecule.

The purinenucleosides and their natural or synthetic analogs are a class of compounds with a broad spectrum of physiological effects. Modification of a single side group at the base or ribose moiety can produce substances with antibiotic, antiviral, or antitumor activity. The biological activity of these analogs can only in part be explained through their different behaviour in the complex purinenucleoside metabolism (1,2). The study of the solution conformations of these derivatives could reveal purely sterical arguments for various modes of biological action of these substances, and might in addition help to understand the complex behaviour of the purinenucleosides in preparative chemistry (3). By application of the rich choice of CW and FTNMR-techniques to this class of compounds the preferred solution conformations can be derived.

Yeast tRNA(Asp) is an elongator tRNA with 4 bases in its variable loop. Few other features characterize its primary structure shown in Figure 1 together with that of yeast tRNA(Phe) (1,2); an exceptionally high GC content, 3 GU and one Gψ base pairs in three of its four stems and an almost self- complementary anticodon (GUC).

The solution conformation of 2'-amino-2'-deoxyadenosine, 3'-amino-3'-deoxyadenosine, and 3'-amino-3'-deoxy-6-N,N-dimethyladenosine have been determined by nuclear magnetic resonance in aqueous and ammonia solutions. The analysis of the ribose moiety is based on the two-state S in equilibrium N model of Altona and Sundaralingam. Longitudinal proton relaxation time and nuclear Overhauser enchancement measurements have been carried out in order to characterize the orientation of the base relative to the ribose. Those studies indicate that 3'-amino-3'-deoxyadenosine and 3'-amino-3'-deoxy-6-N,N-dimethyladenosine exist in solution preferentially in the N-anti-g + conformations. On the other hand, 2'-amino-2'-deoxyadenosine adopts the S-syn-g +/t conformation families. It appears that the base is restricted to the anti conformation in the first two compounds, while in 2'-amino-2'-deoxyadenosine, one third of the molecules in the S state are in the anti range. These studies corroborate the previously proposed correlations between the N state of the ribose and the anti orientation of the base and between the S state of the ribose and the syn orientation of the base.

Radicals produced by X-irradiation at 77 K of 1-methyluracil. HBr crystals have been analysed by electron spin resonance spectroscopy. The results are compared with those obtained on 1-methyluracil crystals. Four radicals have been identified, two of which are present only in 1-methyluracil. HBr crystals: the C6-addition radical and the pyrimidine--anion radical. Warming-up experiments have been performed in order to study the secondary radical reactions. The different mechanisms proposed for the radiolysis of DNA constituents in the solid state are discussed in connection with these findings.

Transfer ribonucleic acids (tRNAs) play a central role in the complex mechanism of protein synthesis. In that process their chief function is to carry amino acids to the ribosomes, to decode the messenger RNA and to incorporate the correct amino acid into the growing polypeptide chain.

Radical formation in single crystals of 1,3-dimethyluracil by X-irradiation has been studied by electron spin resonance at 9.5 GHz. This crystal contains no hydrogen bonds. Only Van der Waals forces are present. Accordingly, after X-irradiation at 300 K, the only radicals observed are those resulting from the excitation path: the H-addition radical at C5 and an H-abstraction radical from a methyl group. Irradiation with light of lambda more than 400 nm induces the transformation of the C5-addition into the C6-addition radical. INDO calculations indicate that the C6-addition radical is protonated at O4. Since this crystal does not contain N-H or O-H bonds, this protonation can only occur through proton-abstraction from a C-H bond of a neighbouring molecule by the carbonyl group. The presence of short contacts between C6 and O4 is taken to suggest that the abstraction occurs at C6.

Different chemical reagents were used to study the tertiary structure of yeast tRNASer, a tRNA with a large variable region: ethylnitrosourea, which alkylates the phosphate groups; dimethylsulphate, which methylates N-7 of guanosine and N-3 of cytosine; and diethylpyrocarbonate, which modifies N-7 of adenine. The non-reactivity of N-3 of cytidine 47:1, 47:6, 47:7 and 47:8 and the reactivity of cytidine 47:3 confirms the existence of a variable stem of four base-pairs and a short variable loop of three residues. For the N-7 positions in purines, accessible residues are G1, G10, Gm18, G19, G30, I34, G35, A36, i6A37, G45, G47, G47:5, G47:9 and G73. The protection of N-7 atoms of residues G9, G15, A21, A22 and G47:9 reflects the tertiary folding. Strong phosphate protection was observed for P8 to P11, P20:1 to P22, P48 to P50 and for P59 and P60. A model was built on a PS300 graphic system on the basis of these data and its stereochemistry refined. While trying to keep most tertiary interactions, we adapted the tertiary folding of the known structures of tRNAAsp and tRNAPhe to the present sequence and solution data. The resulting model has the variable arm not far from the plane of the common L-shaped structure. A generalization of this model to other tRNAs with large variable regions is discussed.

The structure of yeast transfer RNA aspartic acid has been refined in one crystal form to 3 A resolution using the restrained least-squares method of Hendrickson and Konnert and real-space fitting using the FRODO program of Jones. The final crystallographic discrepancy index R is 23.5% for 4585 reflections with magnitudes twice their standard deviations between 10 and 3 A. With lower occupancies for some residues of the D-loop, the phosphate U1, and the base U33, the R-factor is 22.3%. The adaptation of the restrained least-squares program for nucleic acids and the progress of the refinement are described. The conformations are analysed with respect to stereochemistry and folding of the backbone. The contacts and hydrogen bonds of the secondary structure are compared with those of yeast tRNAPhe. The presence of only four bases in the variable loop, instead of five as in yeast tRNAPhe, leads to a rotation of residue 48 and a lateral movement of residue 46. These two rearrangements induce different environments for [U8 . . . A14] . . . A21 as well as for A9 and G45. Otherwise, all tertiary contacts observed in yeast tRNAPhe are present in yeast tRNAAsp, except for the absence of hydrogen-bonding between G18 of the D-loop and C56 of the T-loop. The presence of anticodon triplet pairing leads to a distribution of temperature factors different from that observed in yeast tRNAPhe with a stabilization of the AC stem-and-loop and a destabilization of the T and D-loops. We are inclined to suggest that the labilization of the interactions between the T and D-loops is a consequence of the interaction of the anticodon triplets of symmetry-related molecules through hydrogen bonding, which mimics the interaction between the anticodon and its cognate codon on the messenger RNA.

The ribose conformations of 8-azaadenosine, 8-azaguanosine, and 8-azainosine have been studied using proton magnetic resonance in ND3 solutions, in D2O solutions, and in pyridine solutions. The temperature was varied between - 60 and + 40 ° C in ND3 and between + 10 and + 60 °C in D2O solutions. The analysis is based on the two state S ⇌ N model of the ribose moiety proposed by Altona and Sundaralingam. In D2O, the 8-aza substitution destabilizes the gg rotamer and simultaneously diminishes the population of the S state of the ribose. It is deduced that the anti population of the base is greater in the 8-azapurine (β) ribosides than in the common purine (β) -ribosides.

The importance for macromolecular recognition processes of flexibility localised to backbone segments loops, or macromolecular regions will be stressed. The notion of flexibility used here is derived from crystallographically determined B-values, which depend on the physico-chemical potential surrounding each atom and in which each atom moves. It was shown (1) that the continuous epitopes of tobacco mosaic virus protein (TMVP) correspond to those surface regions in the protein structure which possess a run of high B-values along the polypeptide backbone. In fact, all seven epitopes correspond to local maxima of B-values with only one epitope corresponding to a minor peak. The epitopes were determined by measuring the capacity of natural and synthetic peptides of TMVP to inhibit the reaction between native TMVP and anti-TMVP polyclonal antibodies (2,3). A similar correlation between antigenicity and flexibility was reached with anti-peptide antibodies reacting with a native protein (4). Antigenicity has been also correlated with accessibility (5,6), hydrophilicity (7), and evolutionary variance (8). Because of structural constraints imposed by protein folding on protein structure and protein evolution (hydrophilic regions on the exterior, variations in sequence in external loops and not in the backbone framework), these factors will be associated with flexibility. Besides, other examples of the importance of flexibility in recognition processes can be found.

The three-dimensional structures of two elongator transfer RNAs are known in great details: first that of yeast tRNA(Phe) (1–4) and more recently that of yeast tRNA(Asp) (5–8). As seen in Figure 1, both molecules are folded in an L-shaped conformation, which is also found for initiator tRNAs (9,10). Since the conserved or semi-conserved residues (11) are involved in the tertiary interactions which stabilize this folding (1–10), the L-shaped structure represents the general structural organization of all tRNA molecules. Such structural similarity is satisfactory to explain common functions of tRNAs, namely the interaction with ribosomes, and in the case of elongator tRNAs with elongator factors, but not sufficient to account for specific functions. In that case specific structural features must be involved. Differences in the anticodon sequences account for the decoding of the genetic message on mRNA. Recognition by aminoacyl-tRNA synthetases, which leads to the specific ami noacylation of tRNAs is more complex. Because many aminoacyl-tRNA synthetases recognize isoacceptor tRNAs and catalyze tRNA mischarging (12), these features cannot be simple linear nucleotide sequences, but more likely structural domains found at the three-dimensional level.

Resume Deux structures cristallines de desoxyoligomeres ont ete affinees a haute resolution en utilisant les programmes NUCLIN et NUCLSQ developpes pour l'affinement de l'acide aspartique de levure. L'oligomere de forme B est le dodecamere d(5′OH-C-G-C-G-A-A-T-T-C-G-C-G-3′OH) resolu par Dickerson et ses collaborateurs en 1981; les donnees utilisees ont ete celles deposees a la Brookhaven Data Bank. La structure cristalline de l'hexamere de forme Z d(5′OH-5BrC-G-5BrC-G-5BrC-G-3′OH) a ete faite a Strasbourg. Lors de l'affinement, chaque structure s'est comportee differemment. Plus de soixante molecules d'eau ont ete detectees autour de chaque oligomere. La distribution des molecules de solvant est typique de chaque oligomere. Suite a un probable desordre cristallin, l'hydratation de la forme B n'est pas grande. Le petit sillon de la forme B presente la «colonne vertebrale d'hydratation amplement discutee par Dickerson et ses collaborateurs. L'hydration de la forme Z est la plus complexe dans le sillon caverneux qui correspond au petit sillon. Pour les deux oligomeres, les interactions intermoleculaires ont lieu dans le petit sillon: dans la forme B par l'intermediaire de liaisons hydrogene entre deux guanines; et, dans la forme Z, par l'interaction d'empilement et au travers du reseau du solvant. Le role et l'importance du solvant pour la structure de l'ADN sont discutes a la lumiere de ces resultats.

The crystal structure of yeast tRNAAsp enables visualization of an anticodon-anticodon interaction at the molecular level. Except for differences in the base stacking and twist, the overall conformation of the anticodon loop is quite similar to that of yeast tRNAPhe. The anticodon nucleotide triplets, GUC, of two symmetrically related molecules form a minihelix of the RNA type 11. The modified base m1G37 stacks on both sides of the triplets and enforces the continuity with the anticodon stems. Anticodon association induces long-range conformational changes in the region of the dihydrouracil and thymine loops. Experimental evidence includes the variation in the distribution of temperature factors between yeast tRNAPhe and tRNAAsp, the difference in the self-splitting patterns of tRNAAsp in crystal and solution, and the differential accessibility of cytidines to dimethyl sulfate in free and duplex tRNAAsp. These observations are linked to the fragility and disruption of the G.C Watson-Crick base pair at the corner of the molecule formed by the dihydrouracil and thymine loops.