Figure 2. σ70 trans loading on a σ28-dependent transcription unit in vitro.
(A). Schematic of DNA template carrying Ptar, transcribed-region consensus −10 element (wild-type or mutant) and terminator. Template positions corresponding to pause sites are indicated. Note that the pause sites and terminated transcripts emanating from the Ptar promoter are located one base closer to the transcription start site (+1) than on the λPR′ template (Figure 1A). (See ‘Materials and methods’ for the Ptar promoter sequence.) (B). Analysis of RNA transcripts in vitro. Single-round in vitro transcription reactions were performed with reconstituted RNA polymerase (RNAP) holoenzyme containing σ28 (lanes 1–12), RNAP core enzyme (lanes 13–15) or reconstituted RNAP holoenzyme containing σ70 (lanes 16–18) and three different templates: Ptar with a wild-type (wt) transcribed-region −10 element (lanes 1–6 & 13–15) or a mutated (mut) transcribed-region −10 element (lanes 7–12) and λPR′ with a wild-type transcribed-region −10 element (lanes 16–18). The reactions were performed as a time course with samples taken at 1, 6 and 18 min after transcription was initiated; these reactions were performed in the absence of transcript cleavage factors GreA and GreB, resulting in a characteristic pattern of long-lived pause species (Deighan et al., 2011). Where indicated, excess σ70 (1 μM) was added with the ‘start mix’ after open complex formation. RNAs associated with paused transcription elongation complexes (TECs) (pause) and terminated transcripts (full length) are labeled. The asterisk (*) indicates a shorter terminated transcript that is the result of transcription initiating under the control of the transcribed-region −10 element when the σ70-containing holoenzyme is present in the reaction.