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. 2022 Nov 28;11:e82411. doi: 10.7554/eLife.82411

Figure 6. GlnZ release from the glnA mRNA is necessary for target repression.

(A) RNase E is essential for the processing of glnA mRNA. Escherichia coli ΔglnZ (wild-type [WT]) and ΔglnZ ams-1 (TS) strains harboring pBAD-glnAsal or pBAD-glnAeco were grown to OD600 ~0.5 at 30°C and split into two flasks. The flasks were incubated at either 30°C or 42°C for 30 min and further incubated for 10 min after adding 0.2% l-arabinose. The size is estimated by DynaMarker RNA Low II ssRNA fragment. (B) The nucleotide sequence of RNase E cleavage sites in E. coli K-12 glnA mRNA. The C-terminal amino acid sequence of GlnA is shown below the nucleotide sequence. The glnA stop codon and the GlnZ seed region are boxed. The mutated nucleotides are indicated in red. (C) The processing of GlnZ is abrogated by the mutations in RNase E cleavage sites. GlnZ processed from either the glnA mRNA or the GlnZ precursor sRNA was analyzed by northern blot. 5S rRNA served as a loading control. (D) The processing of GlnZ is required for the repression of SucA in the form of mRNA but not the precursor sRNA. Expression levels of SucA and GlnA were analyzed by western blot. GroEL served as a loading control. E. coli ΔglnZ strains harboring pBAD expression plasmids were grown to exponential phase (OD600 ~1.0) in LB medium in the absence (-) or presence of 0.01% l-arabinose (+). Bar graph represents the fold change of SucA repression by the glnA mRNA induced by arabinose calculated from biological replicates (n>5) with standard deviation. Statistical significance was calculated using one-way ANOVA and denoted as *p<0.05.

Figure 6—source data 1. Figure with the uncropped blots.
Figure 6—source data 2. The original files of the full raw unedited northern blots.
Figure 6—source data 3. Figure with the uncropped blots.
Figure 6—source data 4. The original files of the full raw unedited northern and western blots.

Figure 6.

Figure 6—figure supplement 1. Expression of GlnZ and its targets in Escherichia coli rnc14 mutant.

Figure 6—figure supplement 1.

(A) E. coli wild-type (WT) and rnc14 strains were grown to exponential phase (OD600 ~0.5) in MOPS minimal medium containing 0.2% glucose or 20 mM pyruvate as the carbon source and 0.1% ammonium as the nitrogen source. The expression of AceE, SucA, and SucB, and GroEL was analyzed by western blot. (B) E. coli ΔglnZ (WT) and ΔglnZ rnc14 (rnc14) strains harboring pBAD-glnAeco were grown in LB medium to OD600 ~1.0 at 37°C in the presence or absence of 0.2% l-arabinose. GlnZ was detected by a specific oligonucleotide probe MMO-0419. 5S rRNA was detected by MMO-1056 served as a loading control.