Schematic for DMS treatment, library quality, data analysis, and reporter assays. (A) Generation of DMS-induced reverse transcription (RT) stops in Bacillus subtilis grown at one of four different temperatures. RNA samples are then prepped for Illumina sequencing using Structure-seq2. (B) Comparison of RT stops in three DMS-treated biological replicates grown at 37°C. Pearson correlation coefficients (r) are shown. (C) Schematic representing a 5′ untranslated region (5′ UTR) with unstructured (1 and 3) and structured (2) regions. (D) Reactivity profile of the 5′ UTR from (C) plotted for each temperature. (E) Schematic of RNAT-controlled translation initiation. At low temperatures, the ribosome binding site (RBS) is sequestered, and the ribosome (purple) does not bind. At high temperatures, the RBS is available for the ribosome to bind and initiate translation of the downstream gene.
