tRNA editing by deamination: Balancing affinity and specificity

通过脱氨基进行 tRNA 编辑：平衡亲和力和特异性

• 批准号: 7662426
• 负责人: Juan D Alfonzo
• 金额: $ 30.73万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7662426
• 关键词: Active Sites; Adenosine; Affect; Affinity; Affinity Chromatography; Amino Acids; Anticodon; Architecture; Binding; Biological Assay; C-terminal; Catalysis; Cell Nucleus; Cell physiology; Codon Nucleotides; Cytidine Deaminase; DNA; Deaminase; Deamination; Dependency; Discrimination; Disease; Enzymes; Equilibrium; Escherichia coli; Event; Family member; Fluorescence; Future; Gene Expression; Gene Mutation; Genetic Code; Genetic Screening; In Vitro; Inosine; Kinetics; Knowledge; Lead; Leishmania; Link; Maps; Measures; Mediating; Medical; Methionine; Modification; Nature; Nuclear; Organism; Parasites; Play; Positioning Attribute; Process; Protein Family; Proteins; RNA; RNA Binding; RNA Interference; RNA Recognition Motif; Reaction; Recombinants; Role; Sense Codon; Series; Site; Specific qualifier value; Specificity; Substrate Specificity; System; Testing; Therapeutic Intervention; Threonine-Specific tRNA; To specify; Transfer RNA; Trypanosoma; Trypanosoma brucei brucei; Tryptophan; Variant; base; drug development; flexibility; insight; member; mutant; novel; public health relevance; reconstitution; tRNA adenosine deaminase

DESCRIPTION (provided by applicant): Summary The degeneracy of the genetic code is implied in the need for 61 sense codons to specify 20 different amino acids and, with the exception of methionine and tryptophan, each amino acid is encoded by more than one codon. This discrepancy between codon and amino acid numbers was first explained by Crick's wobble hypothesis, which invoked flexibility between the first anticodon and third codon positions during decoding. Since the inception of the wobble rules, over 100 posttranscriptional modifications have been described with the largest number affecting the anticodon of tRNA. As anticodon modifications accrue, new findings lead to a constant reinterpretation of the wobble rules to include novel effects on tRNA function. Anticodon-sequence alterations that expand decoding capacity are part of a growing number of post-transcriptional changes collectively known as tRNA editing. It is our view that tRNA editing provides a mechanism to effectively accommodate genetic code degeneracy. Editing can also be utilized to regulate gene expression. Furthermore, editing itself can be influenced by the structural context of an editing site and in the case of tRNA can be modulated by posttranscriptional modifications. In this proposal, we have focused on the process of inosine formation in the tRNAs of trypanosomatids. We have discovered the first example of two editing events in a single tRNA, whereby positions 32 and 34 of tRNAThr undergo C to U and A to I editing respectively. The finding that every inosine containing tRNA also undergoes C to U editing at position 32 (5' of the wobble position) raises important questions as to what role the two editing events play in the function of this tRNA. By establishing an in vitro A to I editing assay, we have demonstrated that C to U stimulates A to I editing in vitro. We have also identified the enzyme responsible for A to I editing and shown that a unique feature of this enzyme is its ability to perform two different deamination reactions and also is able to utilize both DNA and RNA as substrates. This proposal will thus focus on answering the specific questions of the nature of the machinery that specifies A to I editing and define how these enzymes achieve their specificity. As an essential step in tRNA maturation in trypanosomatids (Leishmania and Trypanosoma), these types of editing also provide a very attractive target for therapeutic intervention against parasites of very major medical importance. Given the link between tRNA maturation and disease, these studies will further expand our knowledge of the role tRNA processing plays in cellular function. PUBLIC HEALTH RELEVANCE: Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, tRNAs undergo post-transcriptional editing changes that are unique to this system. The enzyme responsible for tRNA editing changes in trypanosomatids possesses substrate specificities that are not shared with any other member of this family of proteins. It is thus important to define what features of these enzymes give them their unique specificity. The propose studies will determine the basis for substrate discrimination of the T. brucei editing enzyme which in the future may open doors towards the development of drugs against this essential activity. These studies will also provide functional and evolutionary insights into important members of the cytidine deaminase (CDA) superfamily.

描述（由申请人提供）：总结遗传密码的退化是暗示的，需要61个感官密码子指定20种不同的氨基酸，除了蛋氨酸和色氨酸外，每个氨基酸都由多个多个密码子编码。 Crick的Wobble假设首先解释了密码子和氨基酸数量之间的这种差异，这在解码过程中调用了第一个反密码子和第三个密码子位置之间的灵活性。自从《摇摆规则》成立以来，已经描述了100多次转录后修改，数量最多影响tRNA的反密码子。随着反登起的修饰，新发现导致对摇摆规则的持续重新解释，以包括对tRNA功能的新作用。扩大解码能力的反登音序列改变是越来越多的转录后变化的一部分，共同被称为tRNA编辑。我们认为，tRNA编辑提供了有效适应遗传代码退化的机制。编辑也可以用于调节基因表达。此外，编辑本身可能会受到编辑位点的结构环境的影响，在tRNA的情况下，可以通过文字后修改来调节。在此提案中，我们专注于锥虫的TRNA中的肌苷形成过程。我们发现了单个tRNA中两个编辑事件的第一个示例，在该trNA中，TRNATHR的32和34位置分别对u进行c和a进行编辑。在第32位（摇摆位置的5'）上，每个包含tRNA的插入的发现也会对C进行C进行编辑，这提出了有关两个编辑事件在该tRNA功能中的作用的重要问题。通过建立一个体外A至I编辑测定，我们已经证明了c to u可以刺激A到我在体外进行编辑。我们还确定了负责A进行A进行编辑的酶，并表明该酶的独特特征是其执行两种不同的脱氨酸反应的能力，并且还能够将DNA和RNA同时用作底物。因此，该提案将着重于回答指定a的机械性质的特定问题，并定义了这些酶如何实现其特异性。作为锥虫（Leishmania和Trypanosoma）tRNA成熟的重要步骤，这些类型的编辑还为对具有非常重要医学重要性的寄生虫的治疗干预提供了非常有吸引力的靶标。鉴于tRNA成熟与疾病之间的联系，这些研究将进一步扩大我们对tRNA处理在细胞功能中起作用的知识。公共卫生相关性：利什曼尼亚属和锥虫属的成员感染了全球数百万的人。在这些生物体中，TRNA经历了该系统独有的转录后编辑更改。负责将锥虫的tRNA编辑变化的酶具有底物特异性，这些特异性与该蛋白质家族的任何其他成员没有共享。因此，重要的是要定义这些酶的特征使它们具有独特的特异性。提出的研究将确定t. brucei编辑酶的底物区分基础，该酶将来可能会打开针对这种基本活动的药物开发的大门。这些研究还将为胞苷脱氨酶（CDA）超家族的重要成员提供功能和进化见解。