Unusual RNA-polymerases: reaction mechanism and substrate specificity of tRNA nucleotidyltransferases

CCA添加酶（tRNA核苷酸转移酶）代表具有不寻常特征的高度特异性RNA聚合酶。它们在tRNA的3 ′端高保真合成C-C-A序列，产生氨酰化位点。虽然它们对CMP和AMP添加具有高度选择性，但这些酶接受细胞内的所有tRNA，在真核生物中甚至是经常显示结构偏差的胞质和线粒体tRNA。除了受体茎外，我们还可以确定第73位是一个重要的识别元件。现在，我们想弄清楚CCA添加酶对这些元素的识别，并了解它们如何影响CCA添加各个步骤的效率和保真度。我们希望扩展我们成功的ESR分析，并表征这些酶的高度有趣的特殊形式中的单个结构域和结构元件的运动，这些酶是特异性识别tRNA和核苷酸所必需的。借助特别纳入的非天然氨基酸，可以交联到相邻的位置，我们希望确定这些酶的分子内相互作用区域是必不可少的functional.In详细的系统发育研究，我们已经确定了非常不寻常的组合不同的tRNA核苷酸转移酶在几个原核生物和真核生物。现在，我们希望表征这些酶并分析它们的特定功能以及它们的底物谱。此外，我们希望使用生物信息学祖先序列重建来识别这些酶的共同祖先，并测试这些酶作为重组蛋白的底物特异性，聚合反应，最佳温度和其他参数。通过这些实验，我们希望澄清是否tRNA核苷酸转移酶与部分活动（CC-和A-添加酶），聚（A）聚合酶或其他亚组的CCA-添加酶代表的祖先状态的演变。

CCA-adding enzymes (tRNA nucleotidyltransferases) represent highly specific RNA polymerases with unusual features. They synthesize the sequence C-C-A at high fidelity at the 3´-end of tRNAs, generating the site of aminoacylation. While they are highly selective for CMP and AMP addition, these enzymes accept all tRNAs within a cell, in eukaryotes even cytosolic and mitochondrial tRNAs that frequently show structural deviations. Besides the acceptor stem, we could identify the discriminator position 73 as an important recognition element. Now we want to clarify the recognition of these elements by CCA-adding enzymes and learn how they influence efficiency and fidelity of the individual steps of CCA-addition. We want to expand our successful ESR analyses and characterize movements of individual domains and structure elements in highly interesting special forms of these enzymes that are required for a specific recognition of tRNA and nucleotides. With the help of specifically incorporated non-natural amino acids that can be cross-linked to neighboring positions, we want to identify intramolecular interacting regions in these enzymes that are essential for functionality.In a detailed phylogenetic study, we have identified highly unusual combinations of different tRNA nucleotidyltransferases in several pro- and eukaryotes. Now we want to characterize these enzymes and analyze their specific functions as well as their substrate spectrum. Furthermore, we want to use bioinformatic ancestral sequence reconstruction to identify common ancestors of such enzymes and to test those as recombinant proteins for substrate specificity, polymerization reaction, temperature optimum and other parameters. With these experiments, we want to clarify whether tRNA nucleotidyltransferases with partial activities (CC- and A-adding enzymes), poly(A) polymerases or other subgroups of CCA-adding enzymes represent the ancestral state in evolution.