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The cytidine at the first anticodon position of archaeal tRNAIle2, which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm2C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNAIle-agm2C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNAIle2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNAIle2 and ATP by the vapour-diffusion method. The crystals of the TiaS-tRNAIle2-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-tRNAIle2-ATP complex, with a Matthews coefficient of 2.8 Å3 Da-1 and a solvent content of 61%.

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