Characterisation of the P<i>aatS</i> promoter.

<p><b>A.</b> Primer extension analysis of the <i>aatS</i> transcript. Lanes 1–4 on the gel are arbitrary size standards, used for calibration, generated by sequencing of M13mp18 phage DNA. Lane 5 shows the primer extension product generated using RNA from wildtype M182 cells carrying the <i>aatS</i>1::<i>lacZ</i> fusion. Lane 6 shows the primer extension product generated using RNA from M182<i>Δcrp</i> cells carrying the <i>aatS</i>1::<i>lacZ</i> fusion. The transcription start site is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157016#pone.0157016.g001" target="_blank">Fig 1B</a>. <b>B.</b> β-galactosidase activity determined using lysates of M182 wildtype or M182Δ<i>crp</i> cells carrying P<i>aatS</i> cloned upstream of <i>lacZ</i> in plasmid pRW50. Values shown are percentages of activity observed in strain M182 (92 Miller units). We obtained 7 and 3 Miller units of activity from lysates of M182 or M182Δ<i>crp</i> carrying promoterless pRW50. Error bars represent the standard deviation of three independent experiments. <b>C.</b> Multi-round i<i>n vitro</i> transcription assay using P<i>aatS</i>. The <i>aatS</i>1 DNA fragment was cloned into pSR upstream of a <i>λoop</i> terminator. Purified, supercoiled pSR plasmid was incubated with purified CRP at 37°C, and the reaction started by the addition of 400 nM σ⁷⁰- RNA polymerase holoenzyme. CRP concentrations are; 0 nM, 200 nM, or 400 nM. The 108 nt RNAI transcript from the pSR replication origin, and the 169 nt transcript from P<i>aatS</i>, are indicated. The gel is calibrated with an arbitrary G+A DNA sequencing reaction as a size standard.