Cryo-EM Reconstructions of E. coli RNAP-σ⁵⁴ Holoenzyme and RNAP with the Crystal Structure of RNAP from Tth Fitted
(A) σ⁵⁴ sequence and functional regions.
(B) Comparison of the RNAP-σ⁵⁴ holoenzyme reconstruction (gray) with RNAP core (green) highlights density regions corresponding to σ⁵⁴.
(C) View from downstream side into DNA binding channel with the α subunit at the bottom. Note the claws on the top and the significantly reduced density of the β′ subunit due to a sequence deletion in E. coli (cyan outlines).
(D) View from the top into the active channel, showing the connecting density between the claws (labeled Db). The asterisk indicates where DNA loading is believed to occur, and σ⁵⁴ densities (black outlines) D1, D2, and D3 are labeled. The orange arrow indicates the location of the σ^A region 3.0 helix.
(E) View from β′ side shows three extra regions of σ⁵⁴ density labeled D1, D2, and D3.
α/α, blue/green; β, magenta; β′, yellow; ω, deep salmon. DS, downstream face; US, upstream face of RNAP relative to promoter DNA. Tth PDB ID code, 1IW7.

Cryo-EM Reconstruction of the Activator-Bound Complex and Comparison with the RNAP-σ⁵⁴ Holoenzyme
(A) RNAP-σ⁵⁴ reconstruction viewed from the β′ side (top) and upstream face (bottom).
(B) Overlay of the RNAP-σ⁵⁴-PspF reconstruction and RNAP-σ⁵⁴ reconstruction, same views as in (A).
(C) RNAP-σ⁵⁴-PspF reconstruction. Crystal structures of PspF (orange) and RNAP-σ^A (as in Figure 1) are fitted. Domain movements in σ⁵⁴ between RNAP-σ⁵⁴ (black labels) and RNAP-σ⁵⁴-PspF (red labels) are indicated.

A Schematic Representation of the Proposed Relative Positions and Movements of σ⁵⁴ Domains and Promoter DNA in the Closed, Intermediate, and Open Complexes
Green, RNAP; blue, σ⁵⁴ domains; red, PspF; orange, DNA.

σ⁵⁴ Domain Assignments
(A) Cryo-EM reconstruction of RNAP in complex with σ⁵⁴ lacking the C-terminal domain (RNAP-σ⁵⁴_1–424) (orange) displays a significantly reduced D3 density compared to the full-length RNAP-σ⁵⁴ reconstruction (gray). D1 and D2 domains are indicated (broken lines).
(B) Location of the σ⁵⁴ C terminus by nanogold labeling. Top panel: class averages of nanogold-labeled RNAP-σ⁵⁴ holoenzyme, with arrows pointing to the gold particles (bright circular spots); middle panel: equivalent reprojections from unlabeled RNAP-σ⁵⁴ holoenzyme reconstruction to those of the corresponding classums (Euler angles indicated at the top) with the positions of gold particles (highest-intensity pixel in classums) marked with a red cross; bottom panel: 3D surface view along the same Euler angles as above with the D3 density regions (yellow circles) and the gold particle positions (red cross) marked. Dashed lines indicate D3 density on the opposite side of the σ⁵⁴ holoenzyme.
(C) The RNAP-σ^A/fork junction DNA model from Taq (PDB ID code 1L9Z) fitted into the holoenzyme reconstruction. The asterisk indicates the location where DNA loads into the RNAP active site. The red cross indicates the center of gold particles.
(D) Comparison of the RNAP-σ⁵⁴ reconstruction (gray) with that of RNAP-σ⁵⁴_52–477 lacking region I (yellow). The density centered around the ^∗ position (where DNA loading occurs) is significantly reduced, suggesting that region I in the RNAP-σ⁵⁴ holoenzyme is located in this area. Broken lines indicate σ⁵⁴ density regions in the σ⁵⁴ truncation mutant reconstructions.

Upstream Promoter DNA Positions in the Holoenzyme and Activator-Bound Intermediate Complex
The site of p-azidophenacyl bromide modification is labeled at the top of the gels, and migration positions of the crosslinked σ⁵⁴ promoter, PspF promoter, and β/β′ promoter species (as identified by immunoblotting the gel with respective antibodies) are indicated.
(A) Denaturing gel showing the crosslinking positions of holoenzyme promoter DNA complexes formed on the S. meliloti nifH promoter.
(B) Side view of the holoenzyme with promoter DNA modeled in orange.
(C) Denaturing gel showing the crosslinking positions of intermediate promoter DNA complexes formed on the same promoter as in (A).
(D) Side view of the bEBP-bound complex with DNA modeled in (yellow). Note the relative shift in DNA (from orange in [B] to yellow in [D]) upon bEBP binding. A simple B-DNA has been used as a model and DNA positions are for indicative purposes only.