The structure of a transcription activation subcomplex reveals how σ(70) is recruited to PhoB promoters

Abstract

PhoB is a two-component response regulator that activates transcription by interacting with the σ(70) subunit of the E. coli RNA polymerase in promoters in which the -35 σ(70)-recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the σ(4) domain of σ(70) fused with the RNA polymerase β subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how σ(4) recognizes the upstream pho box repeat. As with the -35 element, σ(4) achieves this recognition through the N-terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices α2 and α3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost -35 element contacts are compensated by new ones with the activator. In addition, σ(4) is reoriented, thereby suggesting a remodelling mechanism for transcription initiation.

Figure 1

σ₄-β-Flap tip helix chimera binding to DNA. EMSA of the σ₄-β-flap tip helix/PhoB^E/pho box DNA ternary complex. (A) Increasing amounts of σ₄-β-flap tip helix chimera (0, 0.055, 0.11 and 0.18 nmol, respectively) were added to a pre-formed PhoB^E/pho box DNA complex (lanes 2–5) or to a free pho box (lanes 6–8). The positions of the PhoB^E/pho box DNA complex and the σ₄-β-flap/PhoB^E/pho box DNA are indicated (arrow and bracket, respectively). F, free DNA probe. (B) σ₄-β-Flap tip helix interaction with a −35 DNA probe. Increasing amounts of σ₄-β-flap tip helix (0, 0.055, 0.11 and 0.18 nmol, respectively) were added either to pho box DNA (lanes 1–4) or to a DNA probe containing the canonical −35 motif (see Materials and methods). The position of the σ₄-β-flap tip helix/−35 DNA probe is indicated (arrow). F, free DNA probe.

Figure 2

Overview of protein–DNA structures. C^α traces of transcriptional activator PhoB^E (A) and σ₄-β-flap tip helix chimera (B) highlighting important structural elements in different colours.

Figure 3

Electron density maps of the σ₄-β-flap/PhoB^E/pho box DNA complex and details of the σ₄-β-flap tip helix chimera. (A) Experimental σ^A-weighted electron density map of the ternary complex at 6.5 Å after density modification (see Materials and methods), contoured at 1σ. The final model is shown fitted in the density. (B) Final refined σ^A-weighted electron density map of the ternary complex at 4.3 Å. (C) Electrostatic potential surface representation of σ₄ showing the β-flap tip helix as a C^α trace in pale yellow (left and middle panels) in two views 90° apart. The β-flap tip helix fits in a hydrophobic crevice of the σ₄ surface. At the right panel (same view as the middle panel), the electrostatic potential surface representation includes the attached β-flap tip helix.

Figure 4

Structure of the σ₄-β-flap tip helix chimera/PhoB^E/pho box DNA complex. (A) Ribbon diagrams of the ternary complex. The upstream and the downstream protomers of PhoB^E are shown in blue and green, respectively; the coding and template DNA strands are coloured in yellow and orange, respectively; the chimera is shown as σ₄ in red and the β-flap tip helix in pale yellow. Note that the σ₄ subunit interacts only with the upstream PhoB^E. (B, C) Different orientations of the ribbon representation in (B) to show the relative positioning of the different multisubunits of the ternary complex. (D) Alignment of an RNAPH promoter containing the canonical −35 and −10 elements (upper sequence) and the phoA promoter containing the pho box (lower sequence). In the ternary complex reported here, the PhoB^E dimer covers the whole pho box sequence (light violet rectangle), while σ₄ covers the sequence from −35 to −30 (pink rectangle) which coincides with the binding site of σ₄ onto the canonical −35 element (upper sequence, pink rectangle). Two conserved adenines at position −34 in both sequences are highlighted in dark violate squares (see text).

Figure 5

Interactions within the ternary complex. (A) Top: ribbon stereo plot showing the interactions between σ₄ (red) and the pho box DNA. Interacting amino acids and DNA bases are indicated. (B) Stereo plot showing the interactions of σ₄ (red) with the PhoB^E upstream protomer (blue). The view rotated 180° about the horizontal axis relative to (A). (C) Electrostatic potential surface representation of the σ₄ domain bound to the DNA (left) or to the upstream protomer of the PhoB^E tandem (right) within the ternary complex. The σ₄/PhoB^E complex has been ‘opened’ by swinging the σ₄ domain 180° to show the electrostatic potential of the interaction surface. The residues from each subunit that confront the residues from the other subunit in a contacting distance have been indicated. A base from each DNA strand is also shown to make the 180° turn of the structure clearer. The β-flap tip helix (pale yellow) and the downstream PhoB^E protomer (light blue) are shown as C^α traces.

Figure 6

Displacement of σ₄ upon binding to the pho box. (A) Two views of the superposition of the ternary complex reported here and the complex σ₄-DNA (1KU7; Campbell et al, 2002); superposition was done by matching the DNA phosphates from the pho box (our structure) and the −35 element (1KU7). Schematic representations of the σ₄ domains of σ⁷⁰ and σ^A are depicted in red and green, the β-flap tip helix in cream and the PhoB^E dimer in cyan. The σ₄ domain of σ⁷⁰ is partly displaced from the DNA as a result of the presence of PhoB^E. (B) Fitting of the PhoB-DNA and σ₄-β-flap tip helix chimera structure against the RNAPH structure. Colour codes for σ₄, the β-flap tip helix and the PhoB^E dimer are as in (A) for our ternary complex. The σ₄ domain as it is in the RNAPH is shown in blue. RNAPH subunits are differentiated by colours and labelled. The superposition shows that the upstream PhoB^E protomer contacts and reorientates the σ₄ domain. (C) Close-up stereo view of the area indicated as a square in (B). The σ₄ movement would drag the β-flap tip helix, thus facilitating the exit of nascent RNA. The black arrow indicates the movement of the β-flap tip helix, from the position observed in the RNAPH structure (β-flap tip helix in lilac) to the position observed in the present structure, when PhoB^E is bound at the Pho promoter.