Fig. 3: Characterization of the lpxP RNA thermometer.
a, Secondary structure models for the two conformations of the lpxP 5′ UTR as identified by ensemble deconvolution analysis of cold-shocked bacteria, with overlaid in vivo DMS reactivities at 10 °C, along with reactivity profiles and base-pairing probabilities for both conformations. Reactivities are averaged across DH5α and TOP10 cells. The register-shifted SL and SLalt are highlighted in purple. Insets, scatter plot depicting the correlation of base reactivities for the deconvolved conformations across DH5α and TOP10 cells. b, Heat map of pairwise Pearson correlation coefficients (PCC) of normalized DMS reactivities across the two alternative conformations of the lpxP 5′ UTR at 10 °C and the sole 37 °C conformation. c, Structure models for SL and SLalt inferred by phylogenetic analysis. Base pairs showing significant covariation (as determined by R-scape) are boxed in dark green (E < 0.05). Helices showing helix-level covariation support (E < 0.05) are boxed in light green. d, Western blot analysis of SLalt-stabilized mutant expression at 37 °C and 10 °C, 30 min, 1 h and 2 h after IPTG induction and cold shock. LacI was used as the loading control. Analysis is representative of two independent biological replicates. e, Western blot analysis of full-length (5′ UTR + CDS) FLAG-tagged LpxP expression at 10 °C, 1 h after IPTG induction, in wild-type or in Csp single-knockout clones from the KEIO collection. LacI was used as the loading control. Analysis is representative of two independent biological replicates.
