tRNA-dependent Pre-transfer Editing by Prokaryotic Leucyl-tRNA Synthetase
tRNA-dependent Pre-transfer Editing by Prokaryotic Leucyl-tRNA Synthetase
复制标题
原核亮氨酰-tRNA合成酶的tRNA依赖性预转移编辑
DOI：
10.1074/jbc.m109.060616
复制
发表时间：
2010-01-29
影响因子：
4.8
通讯作者：
Wang, En-Duo
中科院分区：
文献类型：
--
作者：
Tan, Min;Zhu, Bin;Wang, En-Duo
为了防止由于氨基酸与蛋白质的错误结合而导致的遗传密码模糊，氨基酰基trna合成酶进化出了编辑活性，以消除中间或最终的非同源产物。本文研究了Ia类亮氨酸- trna合成酶（LeuRS）的不同编辑途径。利用不同的突变和实验条件来破译编辑机制，包括最近开发的靶向LeuRS转移后编辑位点的化合物AN2690。该研究强调了tRNA在转移前和转移后的编辑催化中的重要作用。这两种反应在原核生物风衣水蛭和大肠杆菌leurs中具有相当的效率，尽管大肠杆菌酶倾向于转移后编辑，而风衣水蛭酶倾向于转移前编辑。我们的研究结果还表明，tRNA依赖的预转移编辑严格要求tRNA的cca受体端进入编辑域。令人惊讶的是，这种编辑反应对AN2690具有抗性，AN2690通过在转移后编辑位点与tRNALeu形成共价加合物使酶失活。综上所述，这些数据表明，tRNA在转移后编辑构象中的结合赋予了该酶进行转移前编辑催化的能力，而不管其催化转移后编辑的能力如何。
To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final non-cognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre- and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNALeu in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pre-transfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.