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

生物体被认为保持着很高的翻译准确性，以确保参与细胞基本功能的蛋白质几乎不会发生变化。然而，最近在生命的所有分支中都观察到了一种放松的翻译严格，这表明在某些情况下，生物体可能会容忍比之前估计的更高的翻译错误率。事实上，翻译严格性的降低有可能产生新的蛋白质，提供短期或长期的生存益处。这个项目的总体目标是了解tRNA碱基修改、翻译准确性的变化和细胞适应性之间的联系。研究生和本科生将通过他们在这个项目上的工作接受跨学科的培训。一个进化-行动模块(EvolvingSTEM)已经在新罕布夏州和宾夕法尼亚州的高中实施，并将被介绍给北卡罗来纳州温斯顿-塞勒姆两所公立高中的生物学生。这个项目将检验这样一种假设，即tRNA摆动碱基的修改是翻译的一个可调组成部分，而不是当关闭时会导致错误灾难的触发开关。这项提议有三个目标。分子动力学和酶动力学方法将被用来在体外和硅胶中表征进化的BcTilS：tRNAIle2变体，以了解这些变体中发生的催化和构象变化。TILs：tRNAIle2功能障碍的体内效应，如在AUA密码子上插入哪些氨基酸，tRNAIle2的3‘端存在哪些氨基酸，以及增强适应性的代谢机制将被研究。最后，将确定TIL和tRNAIle2的非规范细胞合作伙伴，这些合作伙伴可能在增强细菌适应性方面发挥替代或额外作用。该奖项反映了NSF的法定使命，并已通过使用基金会的智力优势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 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.