Emerging Roles of Threonylcarbamoyladenosine in Translation & DNA Maintenance

描述（由申请人提供）：由于缺乏对苏氨酰氨甲酰基腺苷（t6 A）生物合成途径的了解，导致在理解tRNA反密码子茎环（ASL）的这种普遍修饰的作用方面存在根本性差距。与ASL的其他修饰一样，预期t6 A在翻译装置的特定组分（如氨酰tRNA合成酶和同源mRNA）识别tRNA中具有关键作用。我们研究的长期目标是追求复杂的tRNA修饰及其生物合成所需的酶的功能的基础知识，并阐明它们在核心细胞过程中的作用。本申请集中于t6 A，重点在于对比真核生物合成途径和原核生物合成途径。核心假设是t6 A酶对翻译和DNA维持都至关重要。该假设源自申请人的实验室最近发现的两种t6 A生物合成酶：Sua 5/YrdC和Kae 1/YgjD家族。初步研究表明，虽然t6 A对酵母中的最佳细胞生长很重要，但两个基因yrdC和ygjD在细菌中是绝对必需的，这表明t6 A是原核生命所必需的。此外，其他实验室的研究表明，这两个家族都与酵母中的端粒维持有关，并且YgjD家族参与维持细菌和线粒体中的DNA完整性。这项研究的基本原理是，一旦这些基因家族在翻译和DNA维持中的作用被理解，它们在原核生物中的重要性及其在端粒维持中的重要性的原因将变得显而易见，这些知识将为抗生素和抗癌靶点领域的新应用打开大门。这一中心假设将通过追求三个具体目标进行测试：1）破译完整的t6 A生物合成途径; 2）确定t6 A在体内翻译中的作用; 3）确定t6 A是否具有与翻译不直接相关的细胞功能。在第一个目标下，候选酶已经被表达并且底物RNA被转录。在第二个目标下，几个双报告质粒，允许测试的作用t6 A的起始和框架的维护已被构建沿着与tRNA微阵列测试的影响t6 A的氨酰化。在第三个目标下，条件本质E。已经构建了在PTet启动子下表达yrdC或ygjD的大肠杆菌菌株，并将其用于抑制子筛选，并且已经开发了鉴定t6 A修饰的其它靶标的策略。这种方法是创新的，因为比较基因组学方法被用来指导实验工作，这项工作揭示了tRNA修饰和DNA维持之间的新联系。这项研究意义重大，因为它将促进我们对这种关键tRNA修饰在基本细胞事件中的作用的理解，并可能揭示新的核心调控机制。

DESCRIPTION (provided by applicant): The lack of knowledge on the biosynthetic pathway leading to threonylcarbamoyladenosine (t6A) has led to a fundamental gap in understanding the role this universal modification of the tRNA anticodon stem loop (ASL). As with other modifications of the ASL, t6A is expected to have critical roles in recognition of tRNAs by the specific components of the translation apparatus such as aminoacyl tRNA synthetases and cognate mRNAs. The long-term goal of our research is to pursue fundamental knowledge on the function of complex tRNA modifications and the enzymes required their biosynthesis, and elucidate their roles in core cellular processes. The current application focuses on t6A with an emphasis on contrasting the eukaryotic and prokaryotic biosynthesis pathways. The central hypothesis is that t6A enzymes are critical for both translation and DNA maintenance. This hypothesis derives from the recent discovery of two t6A biosynthetic enzymes by the applicant's laboratory: the Sua5/YrdC and the Kae1/YgjD families. Preliminary studies show that while t6A is important for optimal cell growth in yeast, the two genes yrdC and ygjD are absolutely essential in bacteria, suggesting that t6A is required for prokaryotic life. In addition, studies by other laboratories have shown that both families are linked to telomere maintenance in yeast and that the YgjD family is involved in maintenance of DNA integrity in bacteria and mitochondria. The rationale for the proposed research is that, once the roles of these gene families in translation and DNA maintenance are understood, the reasons for their essentiality in prokaryotes and their importance in telomere maintenance will become apparent, and together this knowledge will open the door to novel applications in the fields of antibiotics and anticancer targets. This central hypothesis will be tested by pursuing three specific aims: 1) Decipher the complete t6A biosynthetic pathway; 2) Determine the role of t6A in translation in vivo; and 3) Determine if t6A has cellular functions not directly linked to translation. Under the first aim, candidate enzymes have been expressed and substrate RNAs transcribed. Under the second aim, several dual reporter plasmids that allow testing the role t6A in initiation and frame maintenance have been constructed along with tRNA microarrays to test the effect of t6A on aminoacylation. Under the third aim, conditional essential E. coli strains that express yrdC or ygjD under the PTet promoter have been constructed and will be used in suppressor screens and strategies to identify other targets for modification by t6A have been developed. The approach is innovative because comparative genomic methods were used to guide the experimental effort, and this work is revealing new links between tRNA modification and DNA maintenance. The proposed research is significant because it will advance our understanding of the role of this critical tRNA modification in fundamental cellular events and could reveal new core regulatory mechanisms.