Figure 7. Structural basis for 6S RNA specificity for Eσ⁷⁰ over Eσ^S.

(A) Sequence alignment of Eco σ⁷⁰ (RpoD) and σ^S (RpoS). Identical residues are shaded black, homologous residues shaded blue. Eco σ⁷⁰ residues that interact with 6S RNA are denoted by a colored dot underneath. The magenta dots indicate positions that are identical between σ⁷⁰/σ^S. Green dots indicate positions that diverge in sequence. Green dots with red dots inside are positions that were swapped between σ⁷⁰ and σ^S but had no apparent effect on 6S RNA preference (Figure S7). Two loci, denoted in red (σ⁷⁰ K593/H600; σ^S E308/E315), altered 6S RNA preference when swapped (see Figure 4C (Klocko and Wassarman, 2009).

(B) Interaction surfaces for promoter DNA (left; PDB ID 5WT1) (Hubin et al., 2017) and 6S RNA (right) on the σ₄ domain. The RNAP holoenzyme is shown as a molecular surface (β, light cyan; β′, light pink; σ⁷⁰, orange). The surface of σ₄ that contacts the promoter DNA is colored dark yellow (left). Marked with their blue nitrogen atoms are K593 and H600 (using Eco σ⁷⁰ numbering), which are solvent-exposed. On the right, the surface of σ⁷⁰₄ that contacts 6S RNA is colored dark green, illustrating that K593/H600 are engaged with the 6S RNA.

(C) Histogram showing the normalized abortive transcription initiation activity for the indicated σ's (bottom) on the indicated templates (σ⁷⁰-specific promoter T7A1; σ⁷⁰-specific 6S RNA; σ^S-specific promoter DPS). The σ^S-holoenzyme activities are shown normalized with respect to wild-type σ^S, while the σ⁷⁰-holoenzymes are shown normalized to wild-type σ⁷⁰. The relative values of the wild-type σ⁷⁰ and σ^S transcription activities on the different templates (with respect to 6S RNA with σ^S) are listed. The histograms represent the average of at least three independent measurements, the error bars denote S.E.M.s. Mutation of σ^S positions 308 and 315 to the corresponding residues in σ⁷⁰ (σ^S E308K/E315H) increased the relative activity on 6S RNA with less effect on the relative activity on the other templates.

See also Figure S7.