Robo-Therm pipeline for RNA thermometer discovery and validation. (A) The characterized structure and predicted functional motif of a validated RNA thermometer are used as a scaffold to search for similar RNA thermometers. (B) RNArobo search consisting of a map and descriptor based on the characterized secondary structure is performed, with “s” denoting single-stranded regions, “h” denoting helical regions in the structure, “N” denoting any nucleotide, and “*” denoting a nucleotide may or may not be present. (C) RNArobo results are submitted to NCBI Blast and filtered to select candidate thermometers. (D) The secondary structure of the candidate thermometers is predicted via RNAfold (Hofacker 2003). (E) Example of β-galactosidase assay of cells after incubation at 25°C, 37°C, and 42°C used to determine the RNA thermometer activity.
