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.