Figure 4.

Enhanced promoter utilization by Q73W σ²⁸ in vivo and in vitro. (A) Amino acid sequence alignment of Regions 2.3 and 2.4 of E. coli σ⁷⁰ and σ²⁸. Bold letters are residues implicated in promoter melting in σ⁷⁰, and the residues not conserved in σ²⁸ are in gray. The numbers indicate amino acid sequence position. (B) Sequence of σ²⁸-dependent promoter (Ptar) used in this study. Only the wild-type sequence of the tar promoter is shown; promoter variants were constructed from this sequence (Koo et al. 2009b). The native sequence of the tar promoter region is shown in capital letters, vector sequence is in lowercase, and −35 and −10 regions and transcription start site are shown in bold. (C) Expression from tar promoter mutants by RNAP containing wild-type or Q73W σ²⁸ in vivo. β-Galactosidase activities (Miller units) of promoter∷lacZ fusions are shown. Assays were performed in CAG57115 (ΔfliA, ΔflgM) with plasmids expressing σ²⁸ variants and bearing tar promoter variants∷lacZ. All values are averages of three independent experiments. (D) In vivo effects of the Q73W substitution on mutations in σ²⁸ that eliminate promoter recognition determinants. β-Galactosidase activities (Miller units) driven by each σ²⁸ variant on tested promoters are shown. All values are averages of three independent experiments. (Gray bar) Activity of each mutation in wild-type background; (black bar) activity of each mutation in Q73W background. (E) Expression from tar promoter mutants with nonoptimal spacers by RNAP containing wild-type or Q73W σ²⁸ in vivo. β-Galactosidase activities (Miller units) are shown. All values are averages of three independent experiments. The spacer length was varied from 9 to 13 base pairs (bp); the wild-type tar promoter has an 11-bp spacer. (F) Effects of the Q73W substitution on mutations in σ²⁸ that eliminate promoter recognition determinants and of tar promoter variants in vitro. Relative transcription determined from single-round run-off transcription assays (Supplemental Fig. S1) is depicted as bar graphs. Each experiment was repeated a minimum of three times, and numbers above each bar indicate average values of relative transcription. (Left graph) Effect of Q73W substitution on mutations eliminating promoter recognition determinants in σ²⁸. Percent transcription was calculated as (intensity of transcript for RNAP with wild-type or Q73W σ²⁸ carrying the specified additional substitutions in σ²⁸)/(intensity of transcript for RNAP with wild-type or Q73W σ²⁸). (Middle and right graphs) Expression from tar promoters with base substitutions (middle) and nonoptimal spacer lengths (right) by RNAP containing wild-type or Q73W σ²⁸, normalized as described in Figure 3D.