Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization.

Preparation of large quantities of RNA molecules of a defined sequence is a prerequisite for biophysical analysis, and is particularly important to the determination of high-resolution structure by X-ray crystallography. We describe improved methods for the production of multimilligram quantities of homogeneous tRNAs, using a combination of chemical synthesis and enzymatic approaches. Transfer RNA half-molecules with a break in the anticodon loop were chemically synthesized on a preparative scale, ligated enzymatically, and cocrystallized with an aminoacyl-tRNA synthetase, yielding crystals diffracting to 2.4 A resolution. Multimilligram quantities of tRNAs with greatly reduced 3' heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically modified DNA half-molecule templates. This latter approach eliminates the need for large-scale plasmid preparations, and yields synthetase cocrystals diffracting to 2.3 A resolution at much lower RNA:protein stoichiometries than previously required. These two approaches developed for a tRNA-synthetase complex permit the detailed structural study of "atomic-group" mutants.

Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation.

Elongation factor Tu (EF-Tu) binds all elongator aminoacyl-transfer RNAs (aa-tRNAs) for delivery to the ribosome during protein synthesis. Here, we show that EF-Tu binds misacylated tRNAs over a much wider range of affinities than it binds the corresponding correctly acylated tRNAs, suggesting that the protein exhibits considerable specificity for both the amino acid side chain and the tRNA body. The thermodynamic contributions of the amino acid and the tRNA body to the overall binding affinity are independent of each other and compensate for one another when the tRNAs are correctly acylated. Because certain misacylated tRNAs bind EF-Tu significantly more strongly or weakly than cognate aa-tRNAs, EF-Tu may contribute to translational accuracy.

Divalent metal ions and the internal equilibrium of the hammerhead ribozyme.

Thermodynamics of RNA cleavage/ligation were measured for a self-cleaving hammerhead ribozyme in the presence of Ca2+, Co2+, Mg2+, and Mn2+. The internal equilibrium, the ratio of cleaved to ligated RNA, decreases with increasing concentrations of each of the four divalent metal ions in a hyperbolic dependence that shows saturation. The metal ion dependence is not due to changes in ionic strength, and the value of the equilibrium constant at saturation is different for each metal ion. The concentration required to achieve half-saturation of the equilibrium is also different for each metal ion, and the order of apparent metal ion dissociation constants correlates with those measured for dissociation of the same metal ions complexed with tRNA and nucleotides. We interpret the divalent metal ion dependence of the equilibrium in terms of a thermodynamic model invoking noncooperative metal ion dissociation from the cleaved RNA. Thus, at 10 mM Mg2+, a commonly employed condition for hammerhead kinetic studies, metal ion dissociation contributes substantially to the free energy of the equilibrium and drives the hammerhead reaction toward cleaved RNA. Temperature dependencies of the equilibrium reveal that while the entropy and enthalpy changes of the equilibrium depend on the identity of the divalent metal ion, in each case a large entropic driving force overcomes an unfavorable change in enthalpy. This agrees with thermodynamics previously measured for an intermolecular hammerhead in the presence of Mg2+ [Hertel, K. J., & Uhlenbeck, O. C. (1995) Biochemistry 34, 1744-1749].