MOLECULAR MECHANISM OF RNA EDITING ADENOSINE DEAMINASE

RNA编辑是用来描述RNA中核苷酸的结构改变、插入或缺失的术语。如果信使RNA(MRNA)发生这种修饰，可能会导致蛋白质序列的翻译，而不是基因DNA序列预测的蛋白质序列。自从发现了作用于真核细胞核中编码的mRNA的RNA编辑酶后，高等生物的基因序列不能再被假设为完全描述蛋白质序列。因此，RNA编辑在蛋白质表达过程中发生的信息传递的基本过程中发挥着关键作用。此外，从编辑消息中翻译的人类蛋白质(erg.谷氨酸和5-羟色胺受体)与许多神经退行性、精神和行为障碍有关，如中风、癫痫、帕金森氏症和精神分裂症。人类病原体也利用自己的优势进行RNA编辑。丁型肝炎病毒是一种亚病毒人类病原体，在与乙肝病毒同时感染期间会增加患严重肝病的可能性，它需要在终止密码子上编辑RNA，以从同一消息中产生两种蛋白质。这两种蛋白质都是丁型肝炎病毒正确复制和包装所必需的。在许多情况下，一种能够进行特定RNA修饰的酶已经被鉴定和克隆。然而，我们对编辑反应中基本步骤的分子基础的了解，例如底物是如何识别的，令人惊讶地有限。本研究的目的是确定一种RNA编辑型腺苷脱氨酶(ADAR-2)的机制特性。这将通过合成非天然RNA底物类似物并结合酶动力学、凝胶迁移率变化实验和荧光测量对其进行详细分析来实现。在我们的工作之前，还没有一个实验系统来详细、系统地分析RNA编辑机制。这些研究的结果将扩大我们对RNA编辑这一重要过程的基本理解，并最终可能导致控制细胞或病毒RNA及其编码的蛋白质的结构的能力。此外，我们的目标是开发可用于合作的试剂，以阐明ADAR/RNA复合体在反应坐标上的不同点的结构。

RNA editing is a term used to describe the structural alteration, insertion or deletion of nucleotides in RNA. If the modification occurs in messenger RNA (mRNA), it can result in the translation of a protein sequence different from that predicted by the DNA sequence of the gene. Since the discovery of RNA editing enzymes that act on the mRNA encoded in the nuclei of eukaryotic cells, the gene sequences of higher organisms can no longer be assumed to fully describe the protein sequences. Thus, RNA editing plays a pivotal role in the basic process of information transfer that takes place during protein expression. Moreover, human proteins translated from editing messages (erg. glutamate and serotonin receptors) have been implicated in a number of neurodegenerative, psychiatric and behavior disorders such as stroke, epilepsy, Parkinson's disease and schizophrenia. Human pathogens also use RNA editing to their advantage. Hepatitis delta virus, a subviral human pathogen that increases the chance of severe liver disease during concurrent infection with hepatitis B, requires RNA editing at a stop codon to produce two proteins from the same message. Both proteins are required for proper replication and packaging of hepatitis delta virus. In many cases, an enzyme capable of a particular RNA modification has been identified and cloned. However, our understanding of the molecular basis for the fundamental steps in the editing reactions, such as how the substrate is recognized, is surprisingly limited. The goal of this research is to define mechanistic properties of an RNA editing adenosine deaminase (ADAR-2). This will be accomplished through the synthesis of nonnatural RNA substrate analogs and their detailed analysis using a combination of enzyme kinetics, gel mobility shift experiments and fluorescence measurements. Prior to our work, no experimental system existed for a detailed, systematic analysis of RNA editing mechanisms. The results of these studies will extend our basic understanding of the important process of RNA editing and may ultimately lead to the ability to control the structure of cellular or viral RNAs and the proteins they encode. Furthermore, we aim to develop reagents that can be used in collaboration to elucidate the structure of the ADAR/RNA complex at distinct points along the reaction coordinate.