Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization.
2001; National Institutes of Health; Volume: 7; Issue: 11 Linguagem: Inglês
Autores
L.D. Sherlin, T.L. Bullock, Tracy Nissan, John J. Perona, Frederick J. LaRiviere, Olke C. Uhlenbeck, Stephen A. Scaringe,
Tópico(s)Enzyme Structure and Function
ResumoPreparation 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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