Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization

Luke D. Sherlin, Timothy L. Bullock, T. Amar Nissan, John J. Perona*, Frederick J. Lariviere, Olke C. Uhlenbeck, Stephen A. Scaringe

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

99 Scopus citations

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 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" mutants.

Original languageEnglish (US)
Pages (from-to)1671-1678
Number of pages8
JournalRNA
Volume7
Issue number11
StatePublished - 2001

Keywords

  • Aminoacyl-tRNA synthetase
  • RNA ligase
  • T7 RNA polymerase
  • X-ray crystallography

ASJC Scopus subject areas

  • Molecular Biology

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