SHAPE analysis of the RNA thermometer from .

RNA thermometers regulate expression of some genes involved in virulence of pathogenic bacteria such as , , and They often function through temperature-dependent conformational changes that alter accessibility of the ribosome-binding site. The 5'-untranslated region (UTR) of the mRNA from contains a very short RNA thermometer. We have systematically characterized the structure and dynamics of this thermometer at single-nucleotide resolution using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) assays. Our results confirm that the thermometer adopts the predicted hairpin conformation at low temperatures, with conformational change occurring over a physiological temperature regime. Detailed SHAPE melting curves for individual nucleotides suggest that the thermometer unfolds in a cooperative fashion, with nucleotides from both upper and lower portions of the stem gaining flexibility at a common transition temperature. Intriguingly, analysis of an extended 5' UTR sequence revealed not only the presence of the RNA thermometer, but also an additional, stable upstream structure. We generated and analyzed point mutants of the thermometer, revealing elements that modulate its stability, allowing the hairpin to melt under the slightly elevated temperatures experienced during the infection of a warm-blooded host. This work sheds light on structure-function relationships in and related thermometers, and it also illustrates the utility of SHAPE assays for detailed study of RNA thermometer systems.