| Research Abstract |
Threonyl-tRNA synthetase (ThrRS) plays an essential role in protein synthesis by catalyzing aminoacylation of tRNA^<Thr> and by editing misacylation. ThrRS is generally composed of the three domains, an N-terminal editing domain, a catalytic domain and an anticodon-binding domain. In general, an organism possesses one kind of gene for ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in threonylation of tRNA^<Thr> ; one for catalysis and the other for editing of misacylated Ser-tRNA^<Thr>. From sequence alignment of ThrRS-1 and ThrRS-2 with those of other organisms, we have found that the editing domain in ThrRS from archaea are different from those in bacteria and eukaryotes. Furthermore, in several creanarchaea including Aeropyrum pernix K1 and Sulfolobus tokodaii strain 7, each contains two genes encoding either the catalytic
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or the editing domains of ThrRS. In order to clarify the structural basis for the evolutionary divergence, the two types of ThrRSs from crenarchaea Aeropyrum pernix and those from Sulfolobus tokodaii (ApThrRS-1, ApThrRS-2, StThrRS-1 and StThrRS-2) have been overexpressed in Eschericha coli, purified and successfully crystallized by the hanging-drop vapor-diffusion method. Diffraction data were collected, and the structure of selenomethionine-labeled ApThrRS-1 crystal has been solved usingthe MAD method, and the atomic parameters were refined by the least-squares method at 2.3 resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus. The tertiary structure of ThrRS-2 was constructed based on the sequence alignment with similar protein, based on which the dimer structure formed between the two editing domains. Gel shift assay experiments shows that ThrRS-1 and ThrRS-2 do not interact to form a dimer and that each forms a self dimer. Furthermore, we have found that tRNA^<Thr> interacts to both, but ThrRS-1 is strongly bound to tRNA^<Thr>. Finally we succeeded to crystallize the complexes between tRNA^<Thr> and ThrRS-1 and between tRNA and ThrRS-2, respectively. X-Ray analyses of these crystals will reveal the interaction geometry from which the specificities of ThrRS-1 and ThrRS-2 to tRNA^<Thr> will be clarified. Less
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