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.

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.

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.

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.