【作者】 朱斌；

【导师】 王恩多；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2007， 博士

【摘要】 氨基酰-tRNA合成酶(Aminoacyl-tRNA synthetase, AARS)催化tRNA的氨基酰化,生成蛋白质生物合成原料氨基酰-tRNA。为保证遗传密码表达的精确性,相似氨基酸对应的AARS具有编校功能通过水解去除误氨基酰-tRNA。亮氨酰-、异亮氨酰-及缬氨酰-tRNA合成酶(LeuRS, IleRS, ValRS)通过氨基酸转移到tRNA分子上以后的编校,简称“转移后编校”水解错误的氨基酰化产物。这类AARS的编校结构域是插入活性中心称为CP1(Connective Peptide 1)的插入肽段。研究结果表明,来源于原始的超嗜热菌Aquifex aeolicus的LeuRS(AaLeuRS)能编校这一类酶催化产生的氨基酰化产物或误氨基酰化产物,例如Ile-tRNAIle, Val-tRNAIle, Val-tRNAVal, Thr-tRNAVal和Ile-tRNALeu。为了进一步研究AaLeuRS这一广泛的编校活性,设计了携带三重识别元件的RNA小螺旋(minihelixLIV)以模拟原始的tRNA,研究了AaLeuRS,大肠杆菌IleRS和LeuRS,嗜热脂肪芽孢杆菌ValRS单独的CP1肽段编校误氨基酰化小螺旋的能力。结果表明只有AaLeuRS单独的CP1肽段可以水解误氨基酰化的小螺旋,例如Ile-minihelixLIV, Val-minihelixLIV和Thr-minihelixLIV,而IleRS及ValRS的单独的CP1肽段则不能。这些结果说明AaLeuRS可能有着比IleRS及ValRS更为原始的编校特性,其编校结构域可能保留了三种酶共同的祖先编校结构域,它应当具有非专一性的编校功能。研究揭示了AARS催化特异性进化的重要步骤。研究了不同来源的LeuRS的编校途径。对来源于超嗜热菌、大肠杆菌、古细菌、酵母、人胞质和人线粒体的LeuRS的研究结果首次证明:LeuRS总的编校功能可以分为以下三条途径:氨基酸转移到tRNA以前的编校(简称转移前编校),这类编校又分为不依赖和依赖tRNA的转移前编校两种;第三种是转移后编校。来源于真核生物的LeuRS没有明显的依赖tRNA的转移前编校,只有不依赖tRNA的转移前编校,它的活性位点可能位于氨基酰化结构域而非编校结构域。不同的纠错途径偏爱编校不同的相似氨基酸,这种偏爱可受tRNA的调节。这些结果揭示了不同编校途径的意义和进化上的关系。研究了依赖tRNA的转移前和转移后的编校途径,用点突变的方法分离了这两种途径。基于对AaLeuRS的CP1编校结构域保守氨基酸的丙氨酸突变扫描(Ala screen)以及对AaLeuRS突变体氨基酰化和编校功能的生化和结构分析,发现Y358对转移前编校至关重要。Y358A AaLeuRS突变体的编校反应行为直接证明了原核生物LeuRS具有tRNA依赖的转移前编校。将Y358和T269两个位点突变为不同的氨基酸残基,对这些AaLeuRS突变体的研究揭示了依赖tRNA的转移前编校中tRNA和编校结构域相互作用的可能模式;证明虽然Y358在转移前和转移后编校中都发挥重要作用,但作用机制不同。与AaLeuRS的Y358A突变体相反,在T273位点的一系列的点突变体研究中发现,某些突变体,例如T273R,丧失了转移后编校却保留了转移前编校。Y358A和T273R两个突变体可以分别区分转移后编校和依赖tRNA的转移前编校,这将为今后单独研究这两条编校途径的机制提供理想的模型。AaLeuRS编校结构域R322的一系列点突变对编校功能没有影响,却可以抑制酶的氨基酰化功能。动力学研究表明,这些点突变主要影响氨基酰化活性口袋对ATP和氨基酸的结合和催化,亦即影响了氨基酰化活性口袋的构象。进一步实验证明,编校结构域中编校反应的发生会对氨基酰化结构域中的氨基酰化反应产生抑制,这种抑制信号正是通过R322在结构域间传导。编校途径的缺失会加强这种抑制,表明这是一种特殊情况下为保证催化特异性而进行的负反馈抑制机制。对温度敏感型菌株的体内补偿实验支持体外实验的结果。同时我们发现,在编校发生的情况下,R322A和R322K突变反而能回复氨基酰化的能力,这一结果解释了为何某些原核生物LeuRS这一位置上进化为A或K。这些结果揭示了LeuRS为保证催化特异性而进化出的结构域之间的交流机制。研究了AatRNA与合成酶相互作用的结构元件。通过核酸酶探针的足迹保护法、点突变、酶促反应动力学测定等研究方法证明AatRNA上存在不同的识别元件组与LeuRS的不同功能相对应。首次证明tRNALeu保守的反密码子第二位碱基在tRNA依赖的转移前编校反应中是LeuRS重要的识别元件。该结果为揭示tRNA依赖的转移前编校的机制以及LeuRS与tRNALeu的共进化提供了重要线索。更多还原

【Abstract】 Leucyl-, isoleucyl- and valyl-tRNA synthetases form a subgroup of related aminoacyl-tRNA synthetases that catalyze the attachment of similar non-polar amino acids onto their cognate tRNAs. To maintain the fidelity of protein biosynthesis these enzymes also hydrolyze mischarged tRNAs through a post-transfer editing mechanism. Here we show that the ancestralαβ-LeuRS from Aquifex aeolicus and its isolated editing domain (AaLeu-CP1) catalyze the hydrolytic editing of the complete set of aminoacylated tRNAs generated by the three enzymes: Ile-tRNAIle, Val-tRNAIle, Val-tRNAVal, Thr-tRNAVal and Ile-tRNALeu indicating they have nonspecific editing function. A composite minihelixLIV was designed to carry the triple amino acid identity and to mimic the RNA molecule charged by the common ancestor of Leu-, Ile- and ValRS. We found that the three enzymes can aminoacylate minihelixLIV, indicating that they carry remnant-charging activity for this ancient form of transfer RNA. We tested if isolated CP1 domains could hydrolyze these primitive aminoacylated RNAs. We found that only the primitive AaLeu-CP1 could efficiently hydrolyze Ile-, Val- and Thr-minihelixLIV carrying non-cognate amino acids. These data indicate thatA?aLeu-CP1 has retained the hydrolytic function to edit the mischarged RNAs issued from the ancestor of Leu-, Ile- and ValRS. These results support the hypothesis that A. aeolicus -LeuRS is closer than Ile-, and ValRS from the last common ancestor from which it still carries the ambiguous editing function.Leucyl-tRNA synthetase (LeuRS) has editing functions to eliminate misactivated amino acids and mischarged tRNAs for preventing genetic code ambiguity, although the mechanisms are unclear. We studied the LeuRSs from Aquifex aeolicus, Escherichia coli, Pyrococcus horikoshii, Saccharomyces cerevisiae, human cytosol and human mitochondria and separated the total editing of LeuRS into three parts: tRNA-independent pretransfer editing, tRNA-dependent pretransfer editing, and posttransfer editing. The eukaryotic LeuRSs have no obvious tRNA-dependent pretransfer editing. The active site for tRNA-independent pretransfer editing appears to be the aminoacylation site.Leucyl-tRNA synthetase (LeuRS) was believed to have tRNA-dependent editing functions to eliminate misactivated amino acids (pretransfer editing) and mischarged tRNAs (post-transferring editing) for preventing genetic code ambiguity. Although the posttransfer editing had been well characterized, the pretransfer editing remained unclear due to the instability of its substrates and the overlapping of its active site with that of posttransfer editing. In the study, with the comprehensive Ala screen mutagenesis study in the editing domain of Aquifex aeolicus LeuRS (AaLeuRS) we found that the Y358A mutation reduced drastically the pretransfer editing but enhance the posttransfer editing. Based on the known structures we proposed a partial pretransfer editing model including the 3’-terminus of tRNA and Y358, following mutagenesis studies support our model and show that Y358 plays different but important roles in both pre- and posttransfer editing. On the other hand, we took advantage of the subtle difference of the key residue T273 of editing active site in recognition of pre- and posttransfer editing substrates, to produce mutations, typically T273R, which shut down the posttransfer editing without affecting pretransfer editing. In summary, we isolated the two tRNA-dependent editing pathways with the Y358A and T273R AaLeuRS mutants, respectively. And we proposed the possible conformation of 3’-terminus of tRNA in pretransfer editing, which was one of the most doubtful and interesting problems in editing.Mutations of R322 in the AaLeuRS editing domain interrupt the aminoacylation ability of the enzyme. Kinetics analysis indicated that the conformation of aminoacylation active site is affected by the mutations so that the binding and catalysis of the enzyme for ATP and amino acids are affected. The following assays proved that the occurring of editing in the editing domain will suppress the aminoacylation in the aminoacylation domain 30? far off, and the suppressive signal is conducted by R322. The deficiency in editing will enhance the suppression, indicating that the suppression of aminoacylation by editing might be a negative feedback mechanism to guarantee the fidelity of the aminoacylation. These results demonstrated the interdomain communication in LeuRS for the genetic fidelity.AatRNALeu has different binding manners with AaLeuRS as shown by our using enzymatic probe based footprinting study. The absolutely conserved A35 (the second base of anticodon) of AatRNALeu was protected by AaLeuRS in one case but not in the other. Unlike that in other tRNA systems, A35 of AatRNALeu was proved dispensable in aminoacylation reaction. However, the A35U tRNA mutant dramatically reduces the ability to trigger the total editing of AaLeuRS while enhances the posttransfer editing, indicating that A35 is an important recognition element by AaLeuRS in pretransfer editing. These results will improve the understanding of the mechanism of pretransfer editing and the co-evolution of tRNALeu/LeuRS.更多还原