SRP156446 PRJNA484729 Comparison of Listeria monocytogenes 5'' UTR structures To completely understand physiological processes in vivo, it is vital to disclose the conformation of biological molecules inside living cells and correlate their structure with their function. Here, we describe a method, FUSE (5'-UTR Structure Elucidation) that was used to characterize changes in the structures of 5'-UTRs at different conditions inside the human bacterial pathogen Listeria monocytogenes. Using FUSE, we discovered a novel RNA thermoswitch and calculated the ratio of two conformations that this thermoswitch assumes at different conditions. FUSE could also identify the site of base-pairing interaction between a small RNA and mRNA with a single-nucleotide resolution. Strikingly, FUSE discovered a functional mRNA:mRNA interaction, where the expression from an mRNA encoding a chaperone, PrsA2, was stimulated by the direct binding of an mRNA encoding its substrate, LLO, the main Listerial virulence factor. Owing to its focus on 5'-UTRs, FUSE can be applied to analyse such regulatory regions in any living organism.