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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 Å 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 Å 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”
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