Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNA ^Ile2 and ATP

The cytidine at the first anticodon position of archaeal tRNA ^Ile2, which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm ²C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA ^Ile-agm ²C 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 P3221, 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%.