An Songon, Musier-Forsyth Karin
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Biol Chem. 2005 Oct 14;280(41):34465-72. doi: 10.1074/jbc.M507550200. Epub 2005 Aug 8.
Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.
氨酰-tRNA合成酶是多结构域酶，通常具有两种活性以确保翻译准确性。一个合成活性位点催化tRNA氨酰化，而一个编辑活性位点水解错误负载的tRNA。脯氨酰-tRNA合成酶（ProRS）已被证明会将半胱氨酸错误负载到tRNA(Pro)上，但缺乏半胱氨酸特异性编辑功能。最近发现，类似合成酶的流感嗜血杆菌YbaK蛋白可反式水解错误负载的半胱氨酰-tRNA(Pro)。然而，这种单结构域水解酶特异性底物选择的机制尚不清楚。在此，我们证明单独的YbaK似乎缺乏特异性tRNA识别能力。此外，在延伸因子Tu存在的情况下，YbaK无法竞争氨酰-tRNA，这表明YbaK在氨酰-tRNA从合成酶释放之前起作用。支持这一观点的是，交联研究揭示了二元复合物（ProRS.YbaK）和三元复合物（ProRS.YbaK.tRNA）的形成。在不存在和存在tRNA(Pro)的情况下，ProRS与YbaK相互作用的结合常数分别为550 nM和45 nM。这些结果表明，YbaK反式编辑的特异性是通过形成新型ProRS.YbaK.tRNA复合物来确保的。
