Fig 1. The cnfY 5’-UTR comprises a temperature-sensitive regulatory RNA element.
(A) Comparison of cnfY transcript and CNFY protein levels at 25°C and 37°C. Y. pseudotuberculosis YPIII was grown to exponential growth phase (OD600 = 0.5) at 25°C or 37°C and samples were harvested for subsequent qRT-PCR and Western blot analysis. Temperature-dependent transcription was measured by qRT-PCR. Data were analyzed by the ΔΔCt method [30] and cnfY transcription was normalized to that of reference genes nuoB and gyrB. Amounts of cnfY transcript relative to levels detected at 25°C are shown. CNFY signals from Western blot analysis were quantified by integrated density quantification using AlphaEaseFC software. The mean density values and standard deviations from three independent experiments were calculated and normalized by the respective mean density value measured at 25°C. A representative Western blot is displayed in S1A Fig. (B) PARS profiles of the cnfY RNAT (-82 nt to +30 nt from AUG) at 25°C and 37°C. The potential SD sequence, its pairing sequence (anti-SD), and the AUG are marked. (C) PARS-derived secondary structure of cnfY (-82 nt to +30 nt from AUG) at 25°C [18]. The potential SD region and the translation start codon (AUG) are highlighted in black and orange, respectively. (D) The RNA control element in the cnfY 5’-UTR confers temperature-dependent reporter gene expression. To test for temperature-dependent translational control, the cnfY RNAT was translationally fused to bgaB under control of the PBAD promoter (pBO3192). The yscW-lcrF intercistronic region (lcrF RNAT) served as positive control (pBO3146). A schematic representation of the reporter gene fusions is displayed. E. coli DH5α and Y. pseudotuberculosis YPIII cells harboring the corresponding plasmid (technical triplicates per each construct) were grown to an OD600 = 0.5 at 25°C. Afterwards, transcription was induced with 0.01% (w/v) (E. coli) or 0.1% (w/v) (Y. pseudotuberculosis) L-arabinose, respectively. The cultures were split immediately: One half remained at 25°C while the other was transferred into pre-warmed flasks at 37°C. After 30 min incubation, samples were taken for subsequent β-galactosidase assay. The displayed results represent the mean activities from three independent experiments (biological replicates). Mean standard deviations are indicated as error bars.