Organization of an activator-bound RNA polymerase holoenzyme

Abstract

Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor sigma(54) remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with sigma(54) alone, and of RNAP-sigma(54) with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-sigma(54) closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.