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D Moras

Publications and source records attributed to D Moras.

At least 181 records · Page 10Linked to original sources
PubMed1987

Crystal structure of a snake venom cardiotoxin.

Cardiotoxin VII4 from Naja mossambica mossambica crystallizes in space group P61 (a = b = 73.9 A; c = 59.0 A) with two molecules of toxin (molecular mass = 6715 Da) in the asymmetric unit. The structure was solved by using a combination of multiple isomorphous replacement and density modification methods. Model building and least-squares refinement led to an agreement factor of 27% for a data set to 3-A resolution prior to any inclusion of solvent molecules. The topology of the molecule is similar to that found in short and long snake neurotoxins, which block the nicotinic acetylcholine receptor. Major differences occur in the conformation of the central loop, resulting in a change in the concavity of the molecule. Hydrophobic residues are clustered in two distinct areas. The existence of stable dimeric entities in the crystalline state, with the formation of a six-stranded antiparallel beta sheet, may be functionally relevant.

Amino Acid Sequence↗
PubMed1987

Spontaneous assembly of double-stranded helicates from oligobipyridine ligands and copper(I) cations: structure of an inorganic double helix.

Two oligobipyridine ligands containing two and three 2,2'-bipyridine subunits separated by 2-oxapropylene bridges have been synthesized and some of their complexation properties with metal ions have been investigated. In particular, with copper(I) they form, respectively, a dinuclear and a trinuclear complex containing two ligand molecules and two or three Cu(I) ions. In view of the pseudotetrahedral coordination geometry of Cu(I) X bis(bipyridine) sites and of NMR data indicating that the present complexes are chiral, one may assign to these dinuclear and trinuclear species a double-helical structure in which two molecular strands are wrapped around two or three Cu(I) ions, which hold them together. These complexes may thus be termed "double-stranded helicates." Determination of the crystal structure of the trinuclear species has confirmed that it is indeed an inorganic double helix, possessing characteristic features (helical parameters, stacking of bipyridine bases) reminiscent of the DNA double helix. This spontaneous formation of an organized structure by oligobipyridine ligands and suitable metal ions opens ways to the design and study of self-assembling systems presenting cooperativity and regulation features. Various further developments may be envisaged along organic, inorganic, and biochemical lines.

2,2'-Dipyridyl↗
PubMed1987

3-D graphics modelling of the tRNA-like 3'-end of turnip yellow mosaic virus RNA: structural and functional implications.

The tRNA-like structure of the aminoacylatable 3'-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the "pseudoknot". It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3'-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested. Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis. The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA(Val) and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.

Base Sequence↗
PubMed1986

Solvation of the left-handed hexamer d(5BrC-G-5BrC-G-5 BrC-G) in crystals grown at two temperatures.

Crystals of the hexadeoxyoligomer d(5BrC-G-5BrC-G-5BrC-G) were grown at different temperatures (5 degrees C, 18 degrees C and 37 degrees C) in the absence of divalent cations. The crystals grown at 5 degrees C did not diffract X-rays, while those grown at 18 degrees C and 37 degrees C did. The oligomer adopts the left-handed ZI conformation in both crystals. The main difference resides in a more extensive hydration shell in the crystal grown at high temperature than in the crystal grown at low temperature. The high-temperature crystal displays a spine of hydration running deep in the minor groove and linking exocyclic O-2 atoms of the pyrimidine rings. In both crystalline forms, a hydrated sodium ion bound to the N-7 of a guanine ring was found. Strings of water molecules bridging phosphate anionic oxygen atoms are found along the backbone. The absolute values of the propeller-twist are also different in both structures although the values of the twist are very similar. The results point to the importance of the crystallization conditions when analysing fine structural details like solvation properties of oligomers.

Crystallization↗
PubMed1986

Anticodon-anticodon interaction induces conformational changes in tRNA: yeast tRNAAsp, a model for tRNA-mRNA recognition.

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.

Anticodon↗
PubMed1986

Studies on anticodon-anticodon interactions: hemi-protonation of cytosines induces self-pairing through the GCC anticodon of E. coli tRNA-Gly.

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 hemiprotonation 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.

Anticodon↗
PubMed1985

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea.

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.

Alkylation↗
PubMed1985

Crystallographic refinement of yeast aspartic acid transfer RNA.

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.

Anticodon↗
PubMed1985

Crystal structure of yeast tRNAAsp: atomic coordinates.

The atomic coordinates of yeast aspartic acid transfer RNA, as determined from a crystallographic investigation to 3 A resolution, are presented. In the ribose phosphate backbone sugars are in the C(3')-endo pucker, except for residues A7, A9, D16, G17, G18, D19, C20, U48, A58, and U60 which are in the C(2')-endo pucker. A least-squares superposition of the phosphorus atoms of yeast tRNAAsp and yeast tRNAPhe enlightens both an overall structural similarity and significant conformational differences. The largest deviations occur in the D-loop and the anticodon region.

Base Sequence↗
PubMed1985

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA.

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.

Anticodon↗
PubMed1984

Crystallization of transfer ribonucleic acids.

A compilation of crystallization experiments of tRNAs published in literature as well as original results are given and discussed in this paper. Up to now 17 different tRNA species originating from Escherichia coli and from the yeast Saccharomyces cerevisiae have been crystallized. All structural tRNA families are represented, namely the tRNAs with large or small extra-loops and among them the initiator tRNAs. The tRNAs with small variable loops (4 to 5 nucleotides), e.g. tRNAAsp and tRNAPhe, yield the best diffracting crystals. Crystalline polymorphism is a common feature; about 100 different crystal forms have been observed, but only 6 among them enabled structure determination studies by X-ray diffraction. Crystallization strongly depends upon experimental parameters such as the presence of polyamines and magnesium as well as upon the purity and the molecular integrity of the tRNAs. Crystals are usually obtained by vapour diffusion methods using salts (e.g. ammonium sulfate), organic solvents (e.g. isopropanol, dioxane or 2-methyl-2,4-pentane diol) or polyethylene glycol as precipitants. A methodological strategy for crystallyzing new tRNA species is described.

Crystallization↗
PubMed1983

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data.

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Yeast tRNAAsp-aspartyl-tRNA synthetase: the crystalline complex.

Aspartyl-tRNA synthetase from yeast, a dimer of molecular weight 125,000 and its cognate tRNA (Mr = 24,160) were co-crystallized using ammonium sulfate as precipitant agent. The presence in the crystals of both components in the two-to-one stoichiometric ratio was demonstrated by electrophoresis, biological activity assays and crystallographic data. Crystals belong to the cubic space group I432 with cell parameter of 354 A and one complex particle per asymmetric unit. The solvent content of about 78% is favorable for a low resolution structural investigation. By exchanging H2O for D2O in mother liquors, advantage can be taken from contrast variation techniques with neutron radiations. Diffraction data to 20 A resolution were measured at five different contrasts, two of them being close to the theoretical matching point of RNA and protein in the presence of ammonium sulfate. The experimental extinction of the diffracted signal was observed to be close to 36% D2O, significantly different from the predicted value of 41%. The phenomenon can be explained by the existence of a large interface region between the two tRNAs and the enzyme. These parts of the molecules are hidden from the solvent and their protons are less easily exchangeable. Accessibility studies toward chemicals of tRNAAsp in solution and in the presence of synthetase are in agreement with such a model.

Amino Acyl-tRNA Synthetases↗