Open AccessCrystallization and preliminary X‐ray diffraction analysis of an archaeal tRNA‐modification enzyme, TiaS, complexed with tRNA Ile2 and ATP
Author(s) -
Osawa Takuo,
Inanaga Hideko,
Kimura Satoshi,
Terasaka Naohiro,
Suzuki Tsutomu,
Numata Tomoyuki
Publication year - 2011
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309111034890
Subject(s) - transfer rna , crystallization , enzyme , chemistry , x ray crystallography , crystallography , biochemistry , rna , diffraction , physics , organic chemistry , gene , optics
The cytidine at the first anticodon position of archaeal tRNA Ile2 , which decodes the isoleucine AUA codon, is modified to 2‐agmatinylcytidine (agm 2 C) to guarantee the fidelity of protein biosynthesis. This post‐transcriptional modification is catalyzed by tRNA Ile ‐agm 2 C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA Ile2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA Ile2 and ATP by the vapour‐diffusion method. The crystals of the TiaS–tRNA Ile2 –ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P 3 2 21, with unit‐cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS–tRNA Ile2 –ATP complex, with a Matthews coefficient of 2.8 Å 3 Da −1 and a solvent content of 61%.