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. 2020 Sep;26(9):1160–1169. doi: 10.1261/rna.074112.119

FIGURE 1.

FIGURE 1.

The deaD mRNA contains a long 5′ UTR. (A) Schematic description of TSSs upstream of the deaD coding region identified by 5′ RNA-seq. The deaD coding region is depicted by a red rectangle and the upstream region by a red line. Transcription start sites identified by Ettwiller et al. (2016), are denoted by rightward arrows with relative read scores (RRS) indicated at the top. Only sites with RRS > 5 are depicted. Dashed lines and numbers at the bottom indicate the distance between the transcription initiation positions and the start of the coding region. (B) Northern-blot analysis of total RNA isolated from wild-type and ΔdeaD strains. The RNA was analyzed using a radiolabeled riboprobe complementary to the deaD coding region. The deaD mRNA is marked by an arrow. The sizes of molecular weight RNA markers, in nucleotides, run alongside the samples, are shown on the right. A 16S rRNA loading control is also shown. (C) TSS determination by RACE. A reverse transcription product generated using total RNA from a WT strain and a primer complementary to the deaD 5′ UTR was ligated to a linker, amplified, cloned and sequenced. The resulting trace shows the reverse complement sequence at the junction of deaD 5′ UTR and linker sequences (shaded). The sequence of the first ten 5′ nt of the deaD mRNA, starting 838 nt upstream of the start codon, is shown on the right. (D) β-galactosidase assays. Plasmids containing a deaD-lacZ fusion, a derivative that contains mutations in the putative −10 sequence of the deaD promoter, or a 50 bp deletion spanning the −10 and −35 regions, were transformed into a ΔlacZ strain and β-galactosidase activity was measured. Regions that correspond to deaD and lacZ sequences are shown in red and blue, with noncoding and coding regions depicted by lines and rectangles, respectively. β-galactosidase activity was measured after growth at 37°C in LB medium to mid-log phase, in LB medium to stationary phase, in minimal M9 medium to mid-log phase, or after transfer of cultures for 1 h to 16°C following an initial growth to mid-log phase in LB medium at 37°C (cold-shock).