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 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.
Sherlin
L D
LD
Department of Chemistry and Biochemistry, University of California at Santa Barbara, 93106-9510, USA.
Bullock
T L
TL
Nissan
T A
TA
Perona
J J
JJ
Lariviere
F J
FJ
Uhlenbeck
O C
OC
Scaringe
S A
SA
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000138:chemical synthesis* / Q000737:chemistry; D014764:Viral Proteins
D014764:Viral Proteins; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000604:Amino Acyl-tRNA Synthetases