Figure 1. σ70 trans loading on a σ70-dependent transcription unit in vivo (MG1655).
(A) Top: schematic of DNA template carrying λPR', transcribed-region consensus extended –10 element (wild-type or mutant) and terminator (see ‘Materials and methods’ for the λPR′ promoter sequence). Transcribed-region sequences that are complementary to the LNA probe are underlined (grey bar) and the positions corresponding to pause sites are indicated. middle Analysis of RNA transcripts in vivo by LNA probe-hybridization. RNA was isolated from MG1655 cells harvested at an OD600 of 0.8–1.0 (see ‘Materials and methods’). 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; this ratio is expressed as a percentage (relative abundance). Mean and SEM of six independent measurements are shown. Asterisks (*) designate values that were too low (<approximately threefold above background) for accurate quantification. M, 10-nt RNA ladder. bottom Analysis of σ70 levels by Western blot. Amount of soluble σ70 is normalized to the amount in cells carrying the experimental template (wt) and a vector that does not direct σ70 over-production. Mean and SEM of three independent measurements are shown. (B) Top: schematic of DNA template carrying λPR′, initial-transcribed-region σ70-dependent pause element, transcribed-region consensus −10 element and terminator. middle Analysis of RNA transcripts in vivo by locked-nucleic-acid (LNA) probe-hybridization, as in panel A. bottom Analysis of σ70 levels by Western blot.