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.     

Modified nucleosides of Bacillus subtilis transfer ribonucleic acids.

An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented. Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties. Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12). Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine. Therefore, the nucleoside composition of B. subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's. It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.     

Amino acids attached to transfer ribonucleic acid in vivo.

1. tRNA was extracted from rabbit liver by both the phenol and diethyl pyrocarbonate methods under conditions preventing deacylation of the amino acids attached in vivo. 2. After deacylation 12 amino acids were determined by gas-liquid chromatography, by using the flame-ionization and nitrogen-sensitive thermionic detectors. 3. Comparison of the distribution of 12 amino acids attached to tRNA with those contained in total tissue protein and in the free pool showed little correlation. 4. Results for the enzymic charging assay for tRNA in vitro did not correlate satisfactorily with the analysis of amino acids attached to tRNA in vivo. Marked differences were ntoed in comparison made between our own and other published results.