CAREER: Biochemical and Structural Mechanisms Controlling tRNA-Modifying Metalloenzymes

在化学部（CHE）的生命过程化学（CLP）计划和刺激竞争研究的既定计划（EPSCoR）的支持下，来自特拉华州大学的Jeffrey Mugridge正在研究控制金属酶的生化和结构机制，这些金属酶在转移RNA（tRNA）分子上安装了重要的化学修饰。细胞tRNA分子被各种各样的化学修饰修饰所修饰，这些修饰对于正确调整tRNA结构、稳定性以及高效准确的蛋白质合成至关重要。安装tRNA修饰的酶和生化途径的缺陷与从癌症到神经退行性疾病的广泛人类疾病有关。然而，对于许多在tRNA上安装关键化学修饰的酶，人们对这些酶如何选择性地进行修饰反应缺乏清晰的理解。填补这一知识空白对于我们对细胞RNA生物学的基本理解至关重要。该提案将为两类不同的金属依赖性酶（金属酶）定义详细的原子级机制，这些酶在tRNA上进行关键修饰，直接影响蛋白质合成。与这项研究紧密结合，将开发一个基于课程的本科生研究经验（CURE），让来自代表性不足的群体的本科生和高中生参与一个协作的，基于发现的课程，学生将进行和实验测试有关蛋白质-tRNA相互作用的预测。这项工作的结果可以提供新的信息，如何tRNA修饰金属酶进行复杂的，多步反应的tRNA和扩大学生在本科和高中水平的研究为重点的活动的参与。tRNA反密码子环的过度修饰是必要的正确的密码子反密码子识别和mRNA解码的核糖体在翻译过程中。细胞在这些位置安装许多化学复杂的修饰，以确保和控制翻译效率和保真度。该项目结合了生物化学，结构生物学，生物物理学和化学生物学的技术，研究两种不同类型的tRNA修饰金属酶，它们在tRNA的反密码子环上安装顺序修饰。这项工作的目标是：（1）定义这些类别的金属酶安装反密码子环修饰所使用的逐步化学机制，（2）揭示控制tRNA识别和修饰选择性的结构机制和蛋白质构象，以及（3）提供关于这些类别的金属酶如何在细胞中调节的新的，广泛的信息。该研究成果可以加深我们对tRNA生物学的基本理解，并为未来针对这些或类似金属酶和tRNA修饰途径的治疗开发铺平道路。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry (CHE) and the Established Program to Stimulate Competitive Research (EPSCoR), Jeffrey Mugridge from the University of Delaware is studying the biochemical and structural mechanisms controlling metalloenzymes that install important chemical modifications on transfer RNA (tRNA) molecules. Cellular tRNA molecules are decorated with a huge diversity of chemical modifications that are essential for correctly tuning tRNA structure, stability, and efficient and accurate protein synthesis. Defects in the enzymes and biochemical pathways that install tRNA modifications are linked to a wide range of human diseases from cancers to neurodegenerative disorders. However, for many of the enzymes that install key chemical modifications on tRNA, a clear understanding of how these enzymes selectively carry out their modification reactions is missing. Filling this gap in knowledge is important for our fundamental understanding of cellular RNA biology. This proposal will define the detailed atomic-level mechanisms for two distinct classes of metal-dependent enzymes (metalloenzymes) that install key modifications on tRNA that directly impact protein synthesis. Closely integrated with this research, a course-based undergraduate research experience (CURE) will be developed that engages both undergraduates and high-school students from underrepresented groups in a collaborative, discovery-based course where students will make and experimentally test predictions about protein-tRNA interactions. The outcomes from this work could provide new information on how tRNA-modifying metalloenzymes carry out complex, multistep reactions on tRNA and broaden engagement of students in research-focused activities at the undergraduate and high-school levels.Hypermodification of the tRNA anticodon loop is essential for proper codon-anticodon recognition and mRNA decoding in the ribosome during translation. The cell installs numerous, chemically complex modifications at these locations to ensure and control translational efficiency and fidelity. This project combines techniques from biochemistry, structural biology, biophysics, and chemical biology to study two different classes of tRNA-modifying metalloenzymes that install sequential modifications on the anticodon loop of tRNA. The goals of this work are to: (1) define the stepwise chemical mechanisms used by these classes of metalloenzymes to install anticodon loop modifications, (2) unveil the structural mechanisms and protein conformations that control tRNA recognition and modification selectivity, and (3) provide new, broad information about how these classes of metalloenzymes are regulated in the cell. The research outcomes could deepen our basic understanding of tRNA biology and pave the way for future therapeutic development targeting these or similar metalloenzymes and tRNA modification pathways.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.