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 th e determination of high-resolution structure by X-ray crystallography. We d escribe 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 di ffracting to 2.4 Angstrom resolution. Multimilligram quantities of tRNAs wi th greatly reduced 3' heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically modified DNA half-molecule templat es. This latter approach eliminates the need for large-scale plasmid prepar ations, and yields synthetase cocrystals diffracting to 2.3 Angstrom resolu tion at much lower RNA:protein stoichiometries than previously required. Th ese two approaches developed for a tRNA-synthetase complex permit the detai led structural study of "atomic-group" mutants.