FIGURE 3.

The 3′ UTRs of y1235 and y4098 negatively regulate mRNA stability. (A) Schematic representation of y1235, y4098, and their transcriptional terminator (TTS, red line). (B) Relative GFP fluorescence of E. coli cells expressing GFP with the 3′ UTR of y1235 or y4098, or the rrnB terminator, or without an additional 3′ UTR (pAcGFP1). Relative fluorescence intensities were normalized against the OD₆₀₀ value of the bacterial culture, and the fold change in fluorescence was compared with that of the vector control (set as 1). Student’s t-tests were used to calculate p-values. ^∗∗p < 0.001. Means ± SD from three independent experiments are indicated. (C,D) The half-life of y1235 and y4098 mRNAs in WT and Δ3′ UTR mutant strains. Cells were grown at 26°C to an OD₆₀₀ of 0.8, and rifampicin was added at time 0 to block RNA transcription. Samples were removed at time 0, 1 min, 2 min, 4 min, and 6 min. The percentage of residual mRNA at each time-point was measured by qRT-PCR and compared with that at time 0. Dashed lines indicate the time at which half of the detected mRNA remained. mRNA half-life was determined using linear regression analysis and is shown on the dashed lines. Error bars indicate SD of the mean.