The <i>estA2</i> promoter is activated by a Class I CRP dependent mechanism.

Abstract

<p>A) Sequence of the <i>estA2</i> gene regulatory region. The CRP binding site is shown in orange, the UP element is blue and the promoter -10 and -35 elements are shown in purple. The different promoter positions are numbered relative to the transcription start site (+1). B) Location of the P<i>estA2</i> transcription start site. The gel shows the product of an mRNA primer extension analysis to determine the <i>estA2</i> transcription start site (Lane 5). The gel was calibrated using arbitrary size standards (A, C, G and T in Lanes 1–4). C) Binding of CRP to P<i>estA2</i>. The panel shows the result of a DNAse I footprint to monitor binding of CRP to the 93 bp P<i>estA2</i> DNA fragment. The gel is calibrated with a Maxim-Gilbert DNA sequencing reaction. CRP was added at concentrations of 0.35–2.1 µM. D) CRP is required for transcription from P<i>estA2 in vivo</i>. The panel shows a cartoon representation of the 93 bp P<i>estA2::lacZ</i> fusion and a bar chart illustrates LacZ activity in lysates of cells carrying this fusion. Assays were done in LB medium. E) i) Stimulation of P<i>estA2</i> by CRP <i>in vitro</i>. The figure shows the results of an <i>in vitro</i> transcription reaction. The 112 nt transcript initiates from P<i>estA2</i> and the 108 nt RNAI transcript is an internal control. CRP was added at a concentration of 350 nM and RNA polymerase was added at a concentration of 400 nM. ii) quantification of band intensities from the <i>in vitro</i> transcription analysis.</p