Single-molecule analysis of how birth and death of mRNAs are regulated inside a bacterial cell

项目摘要 细菌在我们周围无处不在，在我们的健康和全球生态系统中发挥着关键作用。理解 细菌如何繁殖对保持我们的身体和环境安全和健康至关重要。细菌 可以通过修改它们的基因表达程序来动态地适应各种各样的条件，例如， 通过促进生存所必需的蛋白质的生产，并限制其他蛋白质的浪费生产。 我研究的最终目标是获得对细菌基因调控的丰富理解，因此我研究 转录、翻译和mRNA降解是如何进行的， 在生理环境中调节。由于细菌细胞中没有细胞核， 发生在相同的细胞质体积中，没有明显的分离。因此，细胞机制使 它们在单个细胞内的协调为理解细菌基因提供了重要基础 表达程序。在这里，我描述了我的小组旨在定义可推广原则的项目 细菌中转录、翻译和mRNA降解的时空协调 细胞我们计划回答以下关键问题：（1）转录-翻译的机制是什么 耦合？(2)RNA聚合酶（RNAP）与核糖体之间的相互作用是如何动态调节的？ (3)mRNA降解速率是如何受mRNA年龄和mRNA亚细胞定位的调节的？ 核糖核酸酶E是大肠杆菌中降解mRNA的主要核糖核酸酶吗？我们将回答这些问题， 在活细胞中在单分子水平上成像RNAP、核糖体和RNase E的动力学。结合 这些技术与细菌遗传学，我们将确定因素，可以调节RNAP的动态， 核糖体相互作用，并分析RNase E的定位和功能的亚细胞异质性。 总的来说，我们的工作将揭示细菌基因调控的新机制原则，并产生新的 在单分子水平上测量、控制和模拟基因表达动力学的方法， 活细胞我们的研究结果对广泛的人类健康问题具有潜在的应用价值， 例如促进健康的微生物组，杀死病原体，改善工业流程， 污染

Project Summary Bacteria are everywhere around us, playing critical roles in our health and global ecosystems. Understanding how bacteria thrive is extremely important in keeping our bodies and environments safe and healthy. Bacteria can dynamically adapt to a wide array of conditions by modifying their gene expression program, for example, by boosting the production of proteins necessary for survival and limiting the wasteful production of others. The ultimate goal of my research is to gain an enriched understanding of bacterial gene regulation, thus I study the very fundamental question of how transcription, translation, and mRNA degradation are performed and regulated in physiological settings. Due to the absence of a nucleus in bacterial cells, these three processes occur in the same cytoplasmic volume without clear separations. Therefore, the cellular mechanisms enabling their coordination inside a single cell offer an important foundation for understanding bacterial gene expression programs. Here I describe projects in my group aiming to define the generalizable principles underlying the spatiotemporal coordination of transcription, translation, and mRNA degradation in bacterial cells. We plan to answer the following key questions: (1) What is the mechanism of transcription-translation coupling? (2) How is the interaction between RNA polymerase (RNAP) and ribosome dynamically regulated? (3) How is the rate of mRNA degradation regulated by the age of mRNA and the subcellular localization of RNase E, the major ribonuclease for mRNA degradation in Escherichia coli? We will answer these questions by imaging the dynamics of RNAP, ribosome, and RNase E at the single-molecule level in live cells. Combining these techniques with bacterial genetics, we will identify factors that can modulate the dynamics of RNAP- ribosome interactions and analyze the subcellular heterogeneity in the localization and function of RNase E. Collectively, our work will uncover new mechanistic principles of bacterial gene regulation and generate new methods for measuring, controlling, and modeling gene expression dynamics at the single-molecule level in live cells. The findings from our work have potential applications for a broad range of human health issues, such as promoting healthy microbiomes, killing pathogens, and improving industrial processes to reduce pollution.