The Role of Zinc ions for RNA Binding and Catalytic Function of the DYW-deaminase

项目摘要/摘要 叶绿体转录本的RNA编辑和切割需要具有动态脱氨酶结构域的蛋白质。这个 DYW-脱氨酶具有核苷酸脱氨酶的几个共同特征，并包括 RNA编辑机器的一部分。一些动态脱氨酶蛋白与任何rna都不相关。 编辑功能，是核糖核酸酶切割所必需的。在此基础上，可以将域分解为 一类具有RNA编辑功能，第二类具有核糖核酸酶功能。相关酶 仅作为氨基水解酶发挥作用，对包括RNA编辑在内的各种细胞功能至关重要， 抗体多样性和病毒防御。每个动力脱氨酶结构域与两个锌离子结合。1号锌最有可能是 由所有核苷酸脱氨酶中发现的典型C/HAE-CXXC基序协调。一种独特的C-氨基酸- 末端可能与锌离子配位，位于催化锌离子结合部位附近。一种通用的机制 胞苷脱氨不需要两个锌离子。第二个锌离子可能具有独特的助催化作用 功能，作为结构特征，或在配基结合中起作用。并非所有人都需要锌离子 核糖核酸酶机制及EDTA促进重组人核糖核酸酶切割活性的研究 体外动态脱氨酶。一种或两种锌离子可能作为锌的“开关”来控制脱氨酶。 以及结构域的核糖核酸酶功能。 该项目旨在研究一种模型，在该模型中锌离子充当决定催化作用的“开关”。 功能。将比较具有内切核酸酶和RNA编辑功能的动力脱氨酶中的锌结合 为了研究每个功能类别中是否存在氨基酸差异，以确定锌的化学计量比。一个游泳池 在可能的锌配位残基上进行氨基酸取代的重组Dyw-脱氨酶将是 用来精确定位锌配位残基的位置。核酸内切核酸酶活性将被检测 RDYW-脱氨酶蛋白具有已知的RNA编辑功能和已知的内切核酸酶功能 金属络合剂的存在或不存在。将测量重组蛋白的核糖核酸酶活性 锌的化学计量比和锌配位残基的突变。一系列含氨基的蛋白质 酸变化可能会发现锌结合和酶功能之间的联系，以及建立 RNA裂解的反应机制。一种用于RNA编辑的活性测定，包括添加外源 将开发动态脱氨酶。不同锌含量的动态脱氨酶活性分别为 测定锌离子结合力与氨基水解酶活性的关系。第二个锌 离子可能在配基结合中起着特殊的作用，为了测试这种可能性，核酸配基要求 将使用RNA和带有修饰核苷酸的假定底物来确定Dyw-脱氨酶的活性。 最后，对重组蛋白的结晶条件进行了筛选。晶体在未来可能会被使用 旨在解决动态脱氨酶结构的实验。

Project Summary/Abstract RNA editing and cleavage of chloroplast transcripts require proteins with the DYW-deaminase domain. The DYW-deaminase shares several features common to nucleotide deaminases and comprises the enzymatic portion of the RNA editing machinery. Some DYW-deaminase proteins are not associated with any RNA editing function and are required for ribonuclease cleavage. On this basis the domain can be broken down into one class with RNA editing functions and a second class with ribonuclease functions. Related enzymes function exclusively as amino hydrolases and are critical for various cellular functions including RNA editing, antibody diversity, and viral defense. Each DYW-deaminase domains binds two zinc ions. Zinc #1 is most likely coordinated by the typical C/HAE---CXXC motif found in all nucleotide deaminases. Amino acids in a unique C- terminus likely coordinate Zinc #2 and are nearby the catalytic zinc ion binding site. A general mechanism for cytidine deamination does not require two zinc ions. The second zinc ion could have a unique co-catalytic function, serve as a structural feature, or have a role in ligand binding. Zinc ions are not required for all ribonuclease mechanisms and addition of EDTA promotes the ribonuclease cleavage activity of recombinant DYW-deaminases in vitro. One or both zinc ions might operate as a zinc “switch” that governs the deaminase and ribonuclease functions of the domain. This project aims to investigate a model where zinc ions act as a “switch” that determines catalytic function. Zinc binding in DYW-deaminases with endoribonuclease and RNA editing functions will be compared to investigate if amino acid differences present in each functional classes determine zinc stoichiometry. A pool of recombinant DYW-deaminases with amino acid substitutions in putative zinc coordinating residues will be created to pinpoint the location of zinc coordinating residues. Endoribonuclease activities will be assayed for rDYW-deaminase proteins with known RNA editing functions as well as known endoribonuclease functions in the presence or absence of metal chelators. Ribonuclease activity will be measured for recombinant proteins with altered zinc stoichiometries and mutations in zinc coordinating residues. A series of proteins with amino acid changes might discover a link between zinc binding and enzymatic function as a well as to establish the reaction mechanism for RNA cleavage. An activity assay for RNA editing that includes addition of exogenous DYW-deaminases will be developed. The activity of DYW-deaminases with different zinc stiochiometries will be measured to determine the relationship between zinc ion binding and aminohydrolase activity. The second zinc ion may have a specialized role in ligand binding and to test this possibility the nucleic acid ligand requirements of the DYW-deaminase will be determined using RNAs and putative substrates with modified nucleotides. Finally, crystallization conditions for recombinant proteins will be screened. Crystals could be used in future experiments aimed at solving the structure of the DYW-deaminase.