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）：概述遗传密码的简并性意味着需要61个有义密码子来指定20种不同的氨基酸，并且除甲硫氨酸和色氨酸外，每种氨基酸由一个以上的密码子编码。密码子和氨基酸编号之间的这种差异首先由克里克的摆动假说解释，该假说在解码期间调用第一反密码子和第三密码子位置之间的灵活性。自摆动规则出现以来，已经描述了超过100种转录后修饰，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的转录后变化的一部分，这些变化统称为tRNA编辑。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，编辑本身可以受到编辑位点的结构背景的影响，并且在tRNA的情况下可以通过转录后修饰来调节。在这个建议中，我们集中在锥虫tRNA的肌苷形成的过程。我们已经发现了单个tRNA中两个编辑事件的第一个例子，其中tRNAThr的32和34位分别经历C到U和A到I的编辑。每一个含有次黄苷的tRNA也在32位（摆动位置的5'）经历C到U的编辑，这一发现提出了关于这两个编辑事件在该tRNA的功能中起什么作用的重要问题。通过建立体外A到I编辑测定，我们已经证明了C到U在体外刺激A到I编辑。我们还鉴定了负责A至I编辑的酶，并表明这种酶的独特特征是其能够进行两种不同的脱氨基反应，并且还能够利用DNA和RNA作为底物。因此，该提案将集中于回答指定A到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）中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意义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加工在细胞功能中所起作用的认识。公共卫生相关性：利什曼原虫属和锥虫属的成员感染全世界数百万人。在这些生物体中，tRNA经历转录后编辑变化，这是该系统所独有的。在锥虫中负责tRNA编辑变化的酶具有与该蛋白质家族的任何其他成员不共享的底物特异性。因此，重要的是要确定这些酶的哪些特征赋予它们独特的特异性。这些研究将为T.布鲁氏菌编辑酶，这在未来可能会打开大门，朝着开发药物对这种基本活动。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。