Single-Molecule Study of an RNA Thermosensor

Aurelien Severino, Laura Asensio Puig

Neisseria meningitidis is a human pathogen that has developed subtle mechanisms to evade its host immune system: several genes ensuring its resistance against immune killing are thermoregulated, i.e. increasingly expressed at higher temperature – typically, during host inflammation. The bacterium ability to sense temperature changes is due to special structures occurring in the 5’-UTR region of these genes mRNAs, known as “RNA thermometer” (RNAT). The cssA gene in Neisseria is one of such molecules: its zipper-like structure involves base-pairing of the ribosome binding site (RBS) and therefore hinders ribosome binding at low temperature, whereas at higher temperatures the zipper progressively opens (“melts”), the base-pairing of the RBS is destabilized and the ribosome can bind to the mRNA and synthesize the ’immune evasion’ proteins.

Here, we studied, by means of laser optical tweezers (LOTs), the wild-type (WT) cssA RNAT and a mutant (∆8), having different thermosensing capabilities. Thanks to the LOTs we can get high accuracy data on the behaviour of single molecules, as we can measure forces in the range of pN and distances in the range of nm, while changing temperature. In so-called unzipping experiments, where the molecule is stretched open, we can show that the rupture force of the WT RNAT is higher than that of the ∆8, which is in agreement with the theoretical predictions (MFold) for the sequences. We also observe a high heterogeneity in the unfolding modes of the molecule, with a quasi-continuous low force branch (3-6 pN) featuring two peaks and a high force branch (7-9 pN). The high force peak is in agreement with the predicted structure (rupture force, number of nucleotides). CssA features however many exotic features, such as several misfolded state, a kinetic intermediate, and the fact that it never directly refolds to the native state. The analysis of similar unfolding experiments carried out at different temperatures is currently underway.