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.