Figure 3. Effects of single amino acid substitutions in Region 2.4 and 3.0 of σ²⁸ on activity of the wt tar promoter.

A. Alignment of consensus core −10 and −35 sequences of E. coli σ⁷⁰, σ³² and σ²⁸ promoters. The extended −10 motifs of each promoter are denoted by grey shading.

B. Alignment of amino acid sequence of Regions 2.3, 2.4 and 3.0 of σ⁷⁰, σ³² and σ²⁸. Numbers at each end of sequence indicate amino acid position. Amino acid residues of σ⁷⁰ and σ³² involved in base specific recognition are shown in bold (Koo et al., 2009, Kourennaia et al., 2005, Sanderson et al., 2003, Siegele et al., 1989). Positions of single amino acid substitutions of σ²⁸ used in this study are underlined and the substitutions are shown below the main sequence. The predicted helices at each region was derived from structural data of Thermus aquaticus σ^A and Aquifex aeolicus σ²⁸ (Campbell et al., 2002, Sorenson et al., 2004) and are shown above the sequences.

C. β-galactosidase activity driven by each σ²⁸ variant on the wt tar promoters are shown as a percentage of measured β-galactosidase activity driven by wt σ²⁸. Assay strains are as described in Fig 1B.The different σ²⁸ amino acid substitutions and their locations are shown on the x-axis. All values are averages of three independent experiments; error bars indicate 1 standard deviation.