RNA thermometers regulate expression of some genes involved in virulence of pathogenic bacteria such as Yersinia, Neisseria, and Salmonella. They often function through temperature-dependent conformational changes that alter accessibility of the ribosome-binding site. The 5′-untranslated region (UTR) of the htrA mRNA from Salmonella enterica 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 htrA 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 htrA 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 htrA 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 htrA and related thermometers, and it also illustrates the utility of SHAPE assays for detailed study of RNA thermometer systems.

While visual literacy has been identified as a foundational skill in life science education, there are many challenges in teaching and assessing biomolecular visualization skills. Among these are the lack of consensus about what constitutes competence and limited understanding of student and instructor perceptions of visual literacy tasks. In this study, we administered a set of biomolecular visualization assessments, developed as part of the BioMolViz project, to both students and instructors at multiple institutions and compared their perceptions of task difficulty. We then analyzed our findings using a mixed-methods approach. Quantitative analysis was used to answer the following research questions: (1) Which assessment items exhibit statistically significant disparities or agreements in perceptions of difficulty between instructors and students? (2) Do these perceptions persist when controlling for race/ethnicity and gender? and (3) How does student perception of difficulty relate to performance? Qualitative analysis of open-ended comments was used to identify predominant themes related to visual problem solving. The results show that perceptions of difficulty significantly differ between students and instructors and that students’ performance is a significant predictor of their perception of difficulty. Overall, this study underscores the need to incorporate deliberate instruction in visualization into undergraduate life science curricula to improve student ability in this area. Accordingly, we offer recommendations to promote visual literacy skills in the classroom.