Single-Molecule Insights into the Temperature Dependent Conformational Changes of a RNA Thermometer in the Presence of Crowders and Osmolytes

The helix-coil conformational transition of a ROSE RNA thermometer hairpin unit has been investigated in the presence of salts, osmolytes and macromolecular crowding agents. Using smFRET experiments, we gained new insights into the population of conformational states at the different solution conditions. Whereas an increase of the ionic strength leads to an increased percentage of the closed conformation over a broad temperature range, TMAO, glycine and Ficoll provoke the coexistence of partially unfolded and folded states at low temperature. At high temperatures, TMAO promotes the population of the compact high-FRET state owing to its large excluded volume effect. The results indicate that soft enthalpic interactions between the cosolutes and the RNA hairpin are most likely to cause this destabilizing mode of action. We surmise that in living cells more flexible conformers are preferred to ensure efficient thermosensing, and that the combination of different cellular background molecules enables the cell to regulate the conformational dynamics of its biomolecules by creating the appropriate cellular medium upon demand