Mechanism of proofreading by class I aminoacyl-tRNA synthetases

描述(申请人提供)：氨基酰-tRNA合成酶(AARs)是确保遗传信息在所有活细胞中忠实传递的酶。它们将同源氨基酸与tRNAs配对，通过氨酰-腺苷中间体形成氨基酰-tRNA酯。一些tRNA合成酶不能很准确地区分结构相似的氨基酸，因此不能催化非同源反应。然而，非同源的氨基酰基腺苷和氨基酰基-tRNA被酶的额外水解性破坏(分别在转移前和转移后编辑或校对)。非同源氨基酰化tRNA(转移后编辑)的水解发生在空间分离的编辑结构域中。晶体结构支持这样一种模型，即氨基酰基-tRNA的单链3‘端从合成位置转移到距离约30E的水解位。非同源氨基酰基腺苷的水解(转移前编辑)被认为发生在相同的远程水解点，因此提出了中间体的穿梭。然而，晶体结构从未证实过穿梭路径的存在。最近的研究表明，同源谷氨酰腺苷的水解，类似于转移前编辑的反应，可能发生在非编辑I类谷氨酰tRNA合成酶的活性部位的范围内。这有力地表明，非同源氨基酰腺苷的水解可以发生在I类编辑合成酶的同源合成位点，如异亮氨酰、异亮氨酰和亮氨酰tRNA合成酶(分别为ILERS、ValRS和LEUR)。该项目的长期目标是阐明ILS和ValRS对非同源氨基酸的编辑活动的机制。提出了两个具体目标(1和2)以解决以下问题：(1)转移前编辑是否需要tRNA，如果需要，它的作用是什么？(Ii)非同源氨基酰基腺苷的水解在哪里发生？(Iii)在氨基酰化和/或易位步骤中，氨基酸识别是否通过合成酶和tRNA的构象重新调整来发挥作用？解决这些问题还需要制定适当的方法。一种新的氨基酰基腺苷酸酯合成实验(最近为研究GlnRS而开发)以及新的分配分析方法(本文建议)将被应用于跟踪氨基酰基腺苷酸酯的命运。TRNA类似物和转移后编辑缺陷蛋白突变体将用于实验，旨在评估tRNA的作用和转移前编辑的位置。我们将使用几个不同的荧光团来追踪停流荧光，以探索合成酶和tRNA在非同源反应和同源反应过程中的构象调整。所获得的数据将通过编辑I类氨基酰-tRNA合成酶来显著提高我们对氨基酸识别的理解。与公共健康相关：准确的氨酰-tRNA合成是所有细胞存活的先决条件。因此，氨酰-tRNA合成酶的校对活性通过进化得到了保守，尽管这些反应对细胞施加了很高的能量成本。详细了解编辑机制(特定目标1和2)将允许更好地使用这些酶作为抗菌剂的靶标，因为编辑途径代表了抑制剂作用的另一个选择性靶点。

DESCRIPTION (provided by applicant): Aminoacyl-tRNA synthetases (aaRS) are the enzymes ensuring faithful transmission of genetic information in all living cells. They match cognate amino acids with tRNAs, forming an aminoacyl-tRNA ester by way of an aminoacyl-adenylate intermediate. Some tRNA synthetases cannot distinguish between structurally similar amino acids with high accuracy, and thus proceed in catalysis of the noncognate reaction. However, the noncognate aminoacyl-adenylate and aminoacyl-tRNA are destroyed by additional hydrolytic activities of the enzymes (pre- and post-transfer editing or proofreading, respectively). Hydrolysis of the noncognate aminoacylated tRNA (post-transfer editing) occurs in a spatially separate editing domain. Crystal structures support a model whereby the single stranded 3'-end of the aminoacyl-tRNA is translocated from the synthetic to the hydrolytic site some 30 E distant. Hydrolysis of the noncognate aminoacyl-adenylate (pre-transfer editing) has been assumed to occur at the same remote hydrolytic site and shuttling of the intermediate was therefore proposed. However, crystal structures have never corroborated the existence of the shuttling pathway. Recently, it was shown that hydrolysis of cognate glutaminyl-adenylate, an analogous reaction to pre-transfer editing, may occur in the confines of the active site of a nonediting class I glutaminyl-tRNA synthetase. This strongly suggests that hydrolysis of noncognate aminoacyl-adenylate can occur in the homologous synthetic sites of class I editing synthetases such as isoleucyl-, valyl- and leucyl-tRNA synthetases (IleRS, ValRS and LeuRS, respectively). The long-term objective of this project is to elucidate the mechanisms of the editing activities of IleRS and ValRS toward noncognate amino acids. Two specific aims (1 and 2) are proposed to address the following questions: (i) does pre-transfer editing require tRNA and if it does what is its role? (ii) where does the hydrolysis of noncognate aminoacyl-adenylate take place?; (iii) does amino acid discrimination operate through conformational readjustment of both synthetase and tRNA during aminoacylation and/or translocation step? Addressing these questions will also require the development of suitable methodology. A new aminoacyl-adenylate synthesis assay (recently developed for the study of GlnRS), as well as novel partitioning assays (proposed herein) will be applied to follow the fate of the aminoacyl-adenylate. tRNA analogues and post-transfer editing deficient protein mutants will be used in the experiments designed to evaluate a role for tRNA and location of pre-transfer editing. Stopped flow fluorescence will be pursued, using several different fluorophores, to explore conformational readjustment of synthetase and tRNA during the course of the noncognate and cognate reactions. The data obtained will significantly improve our understanding of amino acid discrimination by editing class I aminoacyl-tRNA synthetases. PUBLIC HEALTH RELEVANCE: Accurate aminoacyl-tRNA synthesis is a prerequisite to the survival of all cells. Consequently, the proofreading activities of aminoacyl-tRNA synthetases have been conserved through evolution in spite of the high energetic cost that the reactions impose on the cell. Understanding of the editing mechanism in detail (Specific Aims 1 and 2) would allow better use of these enzymes as targets for antibacterial agents, because the editing pathway represents an additional selective target for inhibitor action.