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

cca添加酶（tRNA核苷酸转移酶）是具有不同寻常特征的高度特异性RNA聚合酶。它们在trna的3′端高保真地合成C-C-A序列，产生氨基酰化位点。虽然它们对CMP和AMP的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胞质和线粒体trna，这些trna经常表现出结构偏差。除受体干外，鉴别器位置73也是一个重要的识别要素。现在，我们想要澄清这些元素的识别由cca添加酶，并了解它们如何影响效率和保真度的cca添加的各个步骤。我们希望扩展我们成功的ESR分析，并在这些酶的非常有趣的特殊形式中描述单个结构域和结构元件的运动，这些结构域和结构元件是特定识别tRNA和核苷酸所必需的。借助特异性掺入的非天然氨基酸，可以与邻近位置交联，我们希望确定这些酶中对功能至关重要的分子内相互作用区域。在详细的系统发育研究中，我们已经确定了几种原核和真核生物中不同tRNA核苷酸转移酶的高度不寻常的组合。现在我们想要表征这些酶并分析它们的特定功能以及它们的底物光谱。此外，我们希望利用生物信息学的祖先序列重建来确定这些酶的共同祖先，并测试这些酶作为重组蛋白的底物特异性、聚合反应、最适温度和其他参数。通过这些实验，我们想要澄清具有部分活性的tRNA核苷酸转移酶（CC-和A-添加酶）、多聚(A)聚合酶或其他cca添加酶亚群是否代表进化中的祖先状态。

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.