Figure 5

Analysis of the paaL promoter region. (a) Promoter structure of the archetypal σ⁵⁴ factor dependent promoter employed by GenomeMatScan to predict the P. putida KT2440 sigmulon. The upstream activating sequence UAS is indicated, flanked by distal/proximal enhancer binding protein sites displaying diverse spatial positioning upstream of σ⁵⁴-RNA polymerase promoter complex formation. Schematic originally proposed by Cases et al, [38]. (b) Annotated nucleotide sequence of the 456 bp intergenic region between the paaG stop codon, (X), and the paaL start codon (M) in P. putida CA-3. Nucleotide positions are indicated in italics. An imperfect integration host factor (IHF) binding site is highlighted in bold italics with a tetrameric palindrome indicated by directional arrows. Both consensus GG-N₁₀-GC σ⁵⁴ factor binding sites are highlighted in grey, with the primer extension mapped transcriptional start site indicated numerically (+1). (c) RACE directed RT-PCR amplification of the paaL transcriptional start site. Lanes; 1 = 465 bp RACE product, 2 = negative control, (adapter ligated RNA), and M = Hyperladder II DNA marker (Bioline).