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. 2001 Nov;7(11):1671–1678.

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

L D Sherlin 1, T L Bullock 1, T A Nissan 1, J J Perona 1, F J Lariviere 1, O C Uhlenbeck 1, S A Scaringe 1
PMCID: PMC1370207  PMID: 11720294

Abstract

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.

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Selected References

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