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RNA structures are built from recurrent modules that can be identified by structural and comparative sequence analysis. In order to assemble sets of helices in compact architectures, modules that introduce bends and kinks are necessary. Among such modules, kink-turns form an important family that presents sequence and structural characteristics. Here, we describe an internal loop in the bacterial type A RNase P RNA that sets helices bound at the junctions exactly in the same relative positions as in kink-turns but without the structural signatures typical of kink-turns. Our work suggests that identifying a structural module in a subset of RNA sequences constitutes a strategy to identify distinct sequential motifs sharing common structural characteristics.

The hydration patterns around the RNA Watson-Crick and non-Watson-Crick base pairs in crystals are analyzed and described. The results indicate that (i) the base pair hydration is mostly "in-plane"; (ii) eight hydration sites surround the Watson-Crick G-C and A-U base pairs, with five in the deep and three in the shallow groove, an observation which extends the characteristic isostericity of Watson-Crick pairs; (iii) while the hydration around G-C base pairs is well defined, the hydration around A-U base pairs is more diffuse; (iv) the hydration sites close to the phosphate groups are the best defined and the most recurrent ones; (v) a string of water molecules links the two shallow groove 2'-hydroxyl groups, and (vi) the water molecules fit into notches, the size and accessibility of which are almost as important as the number and strength of the hydrophilic groups lining the cavity. Residence times of water molecules at specific hydration sites, inferred from molecular dynamics simulations, are discussed in the light of present data.

Molecular dynamics simulations of macromolecules carrying charges, like nucleic acids, are hampered by proper treatment of the electrostatic problem. Although they can only constitute approximations, dielectric functions depending on the interatomic distance have the advantage of being easier to implement and faster to run than simulations with explicit solvent molecules and periodic boundary conditions. Further, their usefulness has been shown in several instances. Here, we describe a fast algorithm for calculating any dielectric function in MD simulations of biological macromolecules.

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in, virtually, all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids. Here, we present a high-resolution (∼5 A) cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that which causes African Sleeping Sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large eukaryotic expansion segments and ribosomal protein extensions...

Eukaryotic 80S ribosomes of known structure are far more complex than their 70S bacterial counterparts. Those from Saccharomyces cerevisiae, Tetrahymena thermophila, and Triticum aestivum, for example, bear insertions of ribosomal RNA (rRNA) called expansion segments (ES) and additional ribosomal proteins. The ribosomes of the kinetoplastid Trypanosoma brucei, though, are especially fascinating: structurally and their other kinetoplastids’ ribosomes bear very large ESs, as well as smaller ESs, and protein extensions. Additionally, T. brucei ribosomes require novel protein factors for maturation, although they do not require several eukaryotic initiation factors or a recycling factor. As a species, T. brucei is fascinating not only in terms of structure, but also in terms of gene expression and even public health: the species is responsible for the incurable, terminal human African Trypanosomiasis (sleeping sickness); and during post-transcriptional regulation, a single common RNA segment called a splice l...

For the 5-thymyl radical, minor couplings additional to beta-protons bonded to C6 and C7 are observed in single crystals of 1-methylthymine. Hyperfine coupling tensors are given of the N3 nitrogen (+3.5/0.0/0.0 G) and of the exchangeable proton (-4.1/-2.5/-0.9 G) bonded to it. In single crystals of thymine . anhydrate, thymine . monohydrate and 5,6-dihydrothymine these additional couplings are unresolved, but are also present. INDO-calculations were performed to reproduce consistently both beta-coupling constants and additional couplings from N3 and H(N3). Comparison of experimental and calculated values to the conclusion that the 5-thymyl radical is protonated at 04 in all single crystals of thymine derivatives investigated.

H-addition radicals at position C6 of 1-CH3-uracil · HBr and 1-CH3-cytosine · HBr are produced by X-irradiation of single crystals at 300 K. The coupling constants of the methylene β-protons are equivalent at 300 K. Upon cooling they become progressively non-equivalent while a long-range δ coupling appears simultaneously. This additional hyperfine splitting, with an isotropic coupling equal to 4·5–6·5 G, arises from one proton of the methyl group. INDO calculations support the view that the appearance of the long-range proton coupling is correlated with the non-equivalence of the methylene protons. Energy considerations favour a slight asymmetric disposition of the methylene protons relative to the ring, instead of a nonplanar ring, as the cause of the non-equivalence. Symmetry arguments indicate that the non-equivalence of methylene proton coupling is due to close-packing requirements.

Radicals produced at temperatures between 77 K and 300 K by X-irradiation of L-histidine.2HC1 crystals have been analysed by electron spin resonance spectroscopy. The results are compared with previous findings with L-histidine and L-histidine.HC1.H2O crystals. In these three crystals we have identified three main species, which, depending on the molecular environment, are stabilized at different temperatures and produced in different concentrations. These findings are discussed in connection with the observations made on irradiated aqueous solutions of amino acids. It is concluded that our results corroborate the mechanism for the action of ionizing radiation on dry protein proposed by Platzman and Franck (1958)

Radicals produced in X-irradiated single crystals of histidine hydrochloride monohydrate in the temperature range 77–300 K have been investigated using E.S.R. spectroscopy at 9·5 GHz and 35 GHz. The main species trapped at 77 K are the radical anions located at the carboxyl group. These are converted to deamination radicals upon warming to 180 K. Conversion of the anion into the deamination radical is not found if the former is bleached with ultra-violet illumination at 77 K. At room temperature radicals formed by hydrogen addition at the imidazole ring are found after irradiation both at 77 K and 300 K. These have been identified previously. The identifications are supported by MO-SCF calculations at the INDO level. They show that the trapped anion species is represented by the structure RCH(NH2)ĊOOH- rather than RCH(NH3/+)ĊOOH- and the deamination radical by R′CH2ĊHCOOH. The anion radical is characterized by principal values of hyperfine splittings of (20·1, 24·5, 23·0) G for the β proton at the neighbo...

Five different types of radical pairs produced in single crystals of 1-methyl-uracil by irradiation with X-rays were identified. Partial differentiation between pairs was achieved by utilizing different stabilities versus ultraviolet illumination or annealing, and variation of relative yields with temperature. The composition of pairs was derived from hyperfine couplings, the orientation from the maxima of dipolar couplings. These were found to agree with the directional cosines obtained from the crystal structure. Further evidence was provided by half-field spectra (Δm S=2). The zero-field splitting constant D was determined for five pairs, the value of E for one pair. Second-order effects observed at 9·5 GHz and at 35 GHz have been fully analysed for two pairs by application of the appropriate theory which permitted to determine the absolute sign of D. Two pairs are homogeneously composed of hydrogen abstraction radicals 1-methyleneuracil. These are located two layers apart for one pair with the link no...

The complex between cobalt hexammine and decadeoxyoligomer d(CGTACGTACG) crystallizes into the space group P65 with unit cell constants a = b = 17.93A, and c = 43.41A. The molecules have the helix axis coincident with the crystal c-axis. The decamers stack on top of each other and form a quasi-continuous helix. The structure is disordered. The asymmetric unit is a dimer (pPyr-pPur)2 with each base pair 60% of the time a C-G and 40% of the time a T-A. Restrainted least-squares refinement led to an R-factor of 25.5% for 506 observed reflections above the two-sigma level. The structure was found to have one strand in the ZI-conformation and the other in the ZII-conformation. The cobalt hexammine binds to two ZII-chains of symmetrically related molecules. On one ZII chain, two ammonia molecules of the cobalt hexammine bind to the N7 nitrogen and 06 oxygen atoms of the guanine bases and a third ammonia to the phosphate anionic oxygen atom of the preceding pyrimidine base, resulting in an "external" binding mode. On the other ZII chain, one ammonia molecule of the cobalt hexammine binds only to the anionic oxygens of the phosphate group of the guanine bases, leading to an "internal" binding mode. Thus, the basis of the stabilization of Z-DNA by [Co(NH3)6]3+ is its binding to only guanine nucleotides. It is surmised that statistical disordering of deoxyoligonucleotide structures which take a Z conformation, depends on the length of the oligomer. That is to say, octamers and decamers (which cannot use an integral number of molecules for a 12 base pair repeat) form disordered structures whereas tetramers and hexamers form well ordered structures.

SummaryRadical formation in single crystals of hypoxanthine · HCl · H2O, inosine and Na2-5′-IMP · (7·5 H2O) by X-irradiation has been studied using electron-spin-resonance spectroscopy at 9·5 and 35 GHz. In all crystals both H-addition radicals at position C2 and C8 of the purine ring are found. The coupling constants of these two radicals are different and depend strongly on the protonation state of the base. INDO-calculations indicate that the C8-radical is protonated at O6. In Na2-5′-IMP OH-addition radicals at position C2 of the purine ring are formed. Electron adduct radicals are found in the neutral and the N7-protonated base after X-irradiation at 77 K. In Na2-5′-IMP no electron adduct is formed but a radical which probably is the cation. In hypoxanthine · HCl · H2O a radical could be observed after X-irradiation at 77 K, which results from addition of a Cl− to the nitrogen N1.

The radicals produced by X-irradiation at 77 K and at 300 K in cytosine monohydrate crystals have been analysed by electron-spin-resonance (e.s.r.) spectroscopy. Three radicals have been identified at 77 K: the anion radical and the cation radical of the cytosine molecule, together with the radical resutling from H-abstraction from the nitrogen N1. Irradiation at 300 K produces radicals resulting from H-adition at three different positions of the cytosine molecule. These are the C5-addition radical, the C6-addition radical, and the O2-addition radical. The results are compared with those found previously by other authors.

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).

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...