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

基本信息

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
项目类别：
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

项目摘要

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）：总结遗传密码的简并性意味着需要 61 个有义密码子来指定 20 个不同的氨基酸，并且除了甲硫氨酸和色氨酸之外，每个氨基酸都由多个密码子编码。密码子和氨基酸数量之间的这种差异首先由克里克的摆动假说解释，该假说在解码过程中调用了第一反密码子和第三密码子位置之间的灵活性。自摆动规则诞生以来，已有超过 100 种转录后修饰被描述，其中影响 tRNA 反密码子的数量最多。随着反密码子修饰的增加，新的发现导致对摆动规则的不断重新解释，以包括对 tRNA 功能的新影响。扩大解码能力的反密码子序列改变是越来越多统称为 tRNA 编辑的转录后变化的一部分。我们认为，tRNA 编辑提供了一种有效适应遗传密码简并的机制。编辑也可用于调节基因表达。此外，编辑本身可能会受到编辑位点结构背景的影响，并且在 tRNA 的情况下，可以通过转录后修饰进行调节。在本提案中，我们重点关注锥虫 tRNA 中肌苷的形成过程。我们发现了单个 tRNA 中两个编辑事件的第一个例子，tRNAThr 的 32 和 34 位分别经历 C 到 U 和 A 到 I 编辑。每个含有肌苷的 tRNA 也在第 32 位（摆动位置的 5'）经历 C 到 U 编辑，这一发现提出了重要的问题：这两个编辑事件在该 tRNA 的功能中发挥什么作用。通过建立体外 A 至 I 编辑测定，我们证明 C 至 U 可以刺激体外 A 至 I 编辑。我们还鉴定了负责 A 至 I 编辑的酶，并表明该酶的独特特征是其能够执行两种不同的脱氨基反应，并且能够利用 DNA 和 RNA 作为底物。因此，该提案将重点回答指定 A 至 I 编辑的机制的性质的具体问题，并定义这些酶如何实现其特异性。作为锥虫（利什曼原虫和锥虫）tRNA 成熟的重要步骤，这些类型的编辑也为针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目标。鉴于 tRNA 成熟与疾病之间的联系，这些研究将进一步扩展我们对 tRNA 加工在细胞功能中所发挥作用的了解。公共卫生相关性：利什曼原虫属和锥虫属成员感染全世界数百万人。在这些生物体中，tRNA 会经历该系统特有的转录后编辑变化。锥虫中负责 tRNA 编辑变化的酶具有该蛋白质家族的任何其他成员所不具备的底物特异性。因此，定义这些酶的哪些特征赋予它们独特的特异性非常重要。拟议的研究将确定布氏锥虫编辑酶底物辨别的基础，这在未来可能为开发针对这一基本活性的药物打开大门。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方面的见解。