Emerging Roles of Threonylcarbamoyladenosine in Translation & DNA Maintenance

描述（由申请人提供）：由于缺乏导致苏氨基氨基腺苷（t6A）的生物合成途径的知识，导致人们对这种tRNA反密码子干环（ASL）的普遍修饰的作用的理解存在根本性的空白。与ASL的其他修饰一样，t6A有望在翻译装置的特定组分（如氨基酰基tRNA合成酶和同源mrna）识别tRNA方面发挥关键作用。我们的长期研究目标是追求复杂tRNA修饰及其生物合成所需酶的功能基础知识，并阐明其在核心细胞过程中的作用。目前的应用主要集中在t6A，重点是真核生物和原核生物合成途径的对比。核心假设是t6A酶对翻译和DNA维持都至关重要。这一假设源于申请人实验室最近发现的两种t6A生物合成酶：Sua5/YrdC和Kae1/YgjD家族。初步研究表明，虽然t6A对酵母的最佳细胞生长很重要，但yrdC和ygjD两个基因在细菌中是绝对必需的，这表明t6A是原核生物生命所必需的。此外，其他实验室的研究表明，这两个家族都与酵母的端粒维持有关，YgjD家族参与细菌和线粒体DNA完整性的维持。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将变得明显，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来验证：1)破译完整的t6A生物合成途径；2)确定t6A在体内翻译中的作用；3)确定t6A是否具有与翻译不直接相关的细胞功能。在第一个目标下，候选酶被表达，底物rna被转录。在第二个目标下，几个双报告质粒可以测试t6A在起始和框架维持中的作用，并与tRNA微阵列一起构建，以测试t6A对氨基酰化的影响。在第三个目标下，已经构建了在ppet启动子下表达yrdC或ygjD的条件必需大肠杆菌菌株，并将用于抑制子筛选，并且已经开发了识别t6A修饰的其他靶标的策略。这种方法是创新的，因为比较基因组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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the elucidation of a universal tRNA modification pathway essential for accurate protein translation in all kingdoms of life, and for telomere maintenance in eukaryotes, is ultimately expected to reveal novel antibacterial and anticancer targets. Thus, the proposed research is relevant to NIH's mission to support research that increases understanding of life processes and lays the foundation for advances in cancer and/or antibacterial therapeutics.