Riboswitches are regulatory RNA elements that typically act in cis , controlling the expression of their downstream gene in response to changes in metabolite concentration. Interestingly, in Listeria monocytogenes ( Lm ), a saprophyte and opportunistic foodborne pathogen, an S -adenosylmethionine (SAM) riboswitch was previously reported to play a non-canonical trans regulatory role through interaction with an RNA thermosensor (RNAT). The RNAT precedes the mRNA that encodes for positive regulatory factor A (PrfA), a transcriptional activator and the master virulence regulator in Lm . SAM riboswitch element A (SreA) was predicted to bind the prfA RNAT and repress translation of the prfA mRNA, even at elevated temperatures, dampening virulence gene expression. However, the exact molecular determinants of this interaction remain unknown. Using a toolkit of biochemical and biophysical techniques, including electrophoretic mobility shift assays (EMSAs), in vitro transcription/translation assays, and small-angle X-ray scattering (SAXS), we validated this trans interaction. We also designed a minimal SreA construct to identify the sequence and structural elements necessary for binding and translation inhibition. We hypothesize that PrfA translational regulation by SreA occurs co-transcriptionally and results from kinetically driven complex formation. We will further investigate this non-canonical interaction using nuclear magnetic resonance (NMR) spectroscopy, small-angle neutron scattering (SANS), and single-molecule techniques. Our work highlights the critical role non-coding (nc) RNAs play in bacterial gene regulation and their implications for virulence regulation and metabolism.
Wilson et al. (Sun,) studied this question.