Figure 4. Growth phase dependent σ⁷⁰ trans loading on a σ²⁸-dependent transcription unit in vivo.

(A). Detection of RNA transcripts in vivo from the templates shown in Figure 2A by LNA probe-hybridization. Transcribed-region sequences that are complementary to the LNA probe are as in Figure 1A. RNA was isolated from SG110 cells harvested at an OD₆₀₀ of ∼0.5 (log) or ∼2.5 (sta). Pausing is quantified by dividing the signal in the ∼35-nt pause RNA band by the sum of this signal and the signal in the terminated (full-length) band. Mean and SEM of six independent measurements are shown. Asterisks (*) designate values that were too low for accurate quantification. M, 10-nt RNA ladder. (B). top Detection of RNA transcripts derived from the wt template in vivo after treatment with rifampicin. bottom Percent of transcript remaining relative to T = 0 at indicated time points after addition of rifampicin. Mean and SEM of ten (log, 1 m), eight (sta, 1 m), or six (log and sta, 3 m) independent measurements are shown.

DOI: http://dx.doi.org/10.7554/eLife.10514.006

Figure 4—figure supplement 1. (A). Analysis of RNAP-associated transcripts produced from the wild-type Ptar template.

RNA was isolated from the lysate fraction (lys) or the immunoprecipitated fraction (IP) of SG110 cells (OD₆₀₀ ∼2.5) and analyzed by LNA probe-hybridization. The cells contained a vector directing the synthesis of σ²⁸. (B). Analysis of σ⁷⁰ levels by Western blot. Relative quantification of σ⁷⁰ (top) is normalized to the abundance of rpoA (α) in each sample (bottom). Mean and SEM of six independent measurements are shown.

DOI: http://dx.doi.org/10.7554/eLife.10514.007