Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding
Fig 7
Validation of intragenic σ54 promoters for the nagE and yqeB mRNAs.
(A) Intragenic PnagB and PyqeC σ54 promoters were chromosomally mutated. The sequence of both σ54 promoters is shown; conserved, consensus residues (blue text) and the mutagenic changes (red text) are indicated. (B) The relative σ54 occupancy compared to a positive control region (the σ54 promoter of glnA) was measured by ChIP-qPCR at both promoters in wild-type (RPB220 for nagB and RPB232 for yqeB; black bars), ΔPnagB (RPB277; white bars) and ΔPyqeC (RPB279; white bars) E. coli strains. Note that “wild-type” and “mutant” refer to the status of the promoter. Strains used to evaluate PnagB and NagE contain a C-terminal FLAG-tagged nagE gene, whereas the strains used to evaluate PyqeC and YqeB contain a C-terminal FLAG-tagged yqeB gene. (C) Expression of nagE and yqeB, the genes immediately downstream of PnagB and PyqeC σ54 promoters, respectively, relative to the expression of glnA, was measured by RT-qPCR. (D) Western blot probing of extracts from NagE and YqeB FLAG-tagged and untagged E. coli strains with anti-FLAG antibody. Probing the same membranes with anti-α (RNAP subunit) antibody served as a loading control. Wild-type (+) and mutated (-) promoters are indicated. The blot is representative of three independent biological replicates.