Bacteria must sense and respond to their changing environments. In particular, the coordinated expression of virulence genes, proteins that give the bacterium the ability to cause disease, is essential for bacterial fitness. Listeria monocytogenes is a foodborne pathogen that causes severe disease and results in significant public health concerns, economic burden, and societal costs. RNAs play a central regulatory role in gene expression and can sense and respond to the changing host environment, including temperature and metabolite concentrations. Interestingly, two L. monocytogenes RNAs work together to regulate virulence gene expression through an uncharacterized and unconventional mechanism. This project will investigate the RNA-RNA interactions that impact L. monocytogenes virulence gene expression. This work will develop new structural approaches to study RNA structure and conformational rearrangements, and inform RNA molecular recognition and RNA structure-function relationships. Further insights into RNA structure, stability, and intermolecular interactions will aid in engineering of new RNA-based biosensors for biotechnology and generate data for AI-enabled structural models. This project will integrate research and education to foster scientific training and education of high school, undergraduate, and graduate students. Outreach activities are aimed at engaging high school students in full-time research opportunities and preparing them for STEM careers. Furthermore, graduate students will receive training in science communication and develop inquiry-based activities that showcase research to the general public. The overarching scientific objective of this project is to elucidate the structural and molecular mechanisms by which distinct noncoding RNAs in the bacterial pathogen L. monocytogenes interact with each other to regulate virulence gene expression. The research will uncover the non-canonical role of a S-adenosylmethionine sensing riboswitch, Sr