Collaborative Proposal: Role of tRNA base modifications in genetic code accuracy and cellular fitness

生物体被认为保持翻译的高度准确性，以确保在涉及基本细胞功能的蛋白质序列中发生很少的变化。然而，最近在所有生命分支中都观察到一种宽松的翻译严格性，这表明在某些情况下，生物体可能比以前估计的更能容忍翻译错误率。事实上，翻译严格性的降低有可能产生提供短期或长期生存益处的新蛋白质。本项目的总体目标是了解tRNA碱基修饰、翻译精度变化和细胞适应度之间的联系。研究生和本科生将通过他们在这个项目中的工作获得跨学科的训练。一个行动中的进化模块（EvolvingSTEM）已经在新罕布什尔州和宾夕法尼亚州的高中实施，并将在北卡罗来纳州温斯顿-塞勒姆的两所公立高中介绍给生物学学生。该项目将测试tRNA摆动碱基的修饰是翻译的可调组成部分，而不是当关闭时会导致错误灾难的拨动开关的假设。这项提议有三个目标。分子动力学和酶动力学方法将用于在体外和计算机上表征进化的BcTilS:tRNAIle2变体，以了解这些变体中发生的催化和构象变化。研究TilS:tRNAIle2功能障碍的体内效应，如AUA密码子上插入了哪些氨基酸，tRNAIle2的3'端存在哪些氨基酸，以及适应度增强的代谢机制。最后，将鉴定TilS和tRNAIle2的非典型细胞伴侣，它们可能在增强细菌适应性方面发挥替代或额外的作用。该奖项反映了美国国家科学基金会的法定使命，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。

Organisms are presumed to maintain a high accuracy of translation to ensure that little variation occurs in the sequences of proteins involved in essential cellular functions. However, a relaxed translational stringency has recently been observed across all branches of life, suggesting that under certain circumstances organisms may tolerate higher translation error rates than previously estimated. Indeed, a decrease in translation stringency has the potential to yield new proteins that provide short- or long-term survival benefits. The overall objective of this project is to understand the connections between tRNA base modifications, changes in translation accuracy and cellular fitness. Graduate and undergraduate students will receive interdisciplinary training through their work on this project. An evolution-in-action module (EvolvingSTEM) has been implemented in New Hampshire and Pennsylvania high schools and will be introduced to biology students in two Winston-Salem, North Carolina public high schools.This project will test the hypothesis that modification of the tRNA wobble base is a tunable component of translation rather than a toggle switch that when turned off leads to error catastrophe. This proposal has three objectives. Molecular dynamics and enzyme kinetics approaches will be used to characterize evolved BcTilS:tRNAIle2 variants in vitro and in silico in order to understand the catalytic and conformational changes that occur in these variants. The in vivo effects of TilS:tRNAIle2 dysfunction such as which amino acid is inserted at AUA codons, which amino acid is present at the 3'-end of tRNAIle2, and what is the metabolic mechanism of enhanced fitness will be investigated. Finally, noncanonical cellular partners of TilS and tRNAIle2 that may play alternative or additional roles in enhanced bacterial fitness will be identified.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.