E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

Abstract

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ⁷⁰ region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

Keywords: E. coli; RNA polymerase; TraR; biochemistry; chemical biology; cryo-electron microscopy; molecular biophysics; structural biology; transcription initiation.

Figure 1.. Cryo-EM structure of TraR-Eσ⁷⁰.

(top) Color-coding key. (A) TraR-Eσ⁷⁰(I) - cryo-EM density map (3.7 Å nominal resolution, low-pass filtered to the local resolution) is shown as a transparent surface and colored according to the key. The final model is superimposed. (B) TraR-Eσ⁷⁰(II) - cryo-EM density map (3.8 Å nominal resolution, low-pass filtered to the local resolution) is shown as a transparent surface and colored according to the key. The final model is superimposed. (C) Top view of TraR-Eσ⁷⁰(I). The boxed area is magnified in (D). (D) Magnified top view of TraR-Eσ⁷⁰(I) - shows TraR_N (starting near RNAP active site Mg²⁺, extending out secondary channel), TraR_G (interacting primarily with β'rim-helices), and TraR_C (interacting with βlobe-Si1). (E – G) Cryo-EM density (blue mesh) defining the TraR structure. (E) TraR_N and -NADFDGD- motif of RNAP β' (chelating active site Mg²⁺). (F) TraR_G. (G) TraR _C.

Figure 1—figure supplement 1.. Cryo-EM solution conditions do not affect TraR function and TraR-Eσ⁷⁰ cryo-EM processing pipeline.

(A) Multi-round in vitro transcription of rpsT P2 by Eσ⁷⁰ (20 nM) at a range of TraR concentrations (wedge indicates 4 nM - 4 µM) in the absence or presence of 8 mM CHAPSO as indicated. Plasmid templates also contained the RNA-1 promoter. Small effects of TraR on RNA-1 have been reported previously (Gopalkrishnan et al., 2017) but the physiological significance is unclear. (B) Quantification of transcripts from experiments like those in (A) plotted relative to values in the absence of TraR. The IC₅₀ for inhibition by TraR was ~50 nM for both ± CHAPSO data sets. Averages with range from two independent experiments are plotted. (C) Transcription in the absence of TraR is plotted, relative to the same reactions without CHAPSO. Although it had no effect on the concentration of TraR required for half-maximal inhibition (B), CHAPSO reduced transcription slightly. Averages with range from two independent experiments are plotted. (D) TraR-Eσ⁷⁰ cryo-EM processing pipeline.

Figure 1—figure supplement 2.. TraR-Eσ⁷⁰ cryo-EM.

(A) Representative micrograph of TraR-Eσ⁷⁰ in vitreous ice. (B) The ten most populated classes from 2D classification. (C) Angular distribution for TraR-Eσ⁷⁰(I) particle projections. (D) Angular distribution for TraR-Eσ⁷⁰(II) particle projections. (E) (top) The 3.7 Å resolution cryo-EM density map of TraR-Eσ⁷⁰(I) is colored according to the key. The right view is a cross-section of the left view. (bottom) Same views as (top) but colored by local resolution (Cardone et al., 2013). (F) (top) The 3.8 Å resolution cryo-EM density map of TraR-Eσ⁷⁰(II) is colored according to the key. The right view is a cross-section of the left view. (bottom) Same views as (top) but colored by local resolution (Cardone et al., 2013). (G) Gold-standard FSC of TraR-Eσ⁷⁰(I). The gold-standard FSC was calculated by comparing the two independently determined half-maps from RELION. The dotted line represents the 0.143 FSC cutoff, which indicates a nominal resolution of 3.7 Å. (H) FSC calculated between the refined structure and the half map used for refinement (work), the other half map (free), and the full map. (I) Gold-standard FSC of TraR-Eσ⁷⁰(II). The gold-standard FSC was calculated by comparing the two independently determined half-maps from RELION. The dotted line represents the 0.143 FSC cutoff, which indicates a nominal resolution of 3.8 Å. (J) FSC calculated between the refined structure and the half map used for refinement (work), the other half map (free), and the full map.

Figure 1—figure supplement 3.. Eσ⁷⁰ cryo-EM processing pipeline.

Eσ⁷⁰ cryo-EM processing pipeline.

Figure 1—figure supplement 4.. Eσ⁷⁰ cryo-EM.

(A) Representative micrograph of Eσ⁷⁰ in vitreous ice. (B) The ten most populated classes from 2D classification. (C) Angular distribution for Eσ⁷⁰ particle projections. (D) The 4.1 Å resolution cryo-EM density map of Eσ⁷⁰ is colored according to the key. The right view is a cross-section of the left view. (E) Same views as (D) but colored by local resolution (Cardone et al., 2013). (F) Gold-standard FSC of Eσ⁷⁰. The gold-standard FSC was calculated by comparing the two independently determined half-maps from RELION. The dotted line represents the 0.143 FSC cutoff, which indicates a nominal resolution of 4.1 Å. (G) FSC calculated between the refined structure and the half map used for refinement (work), the other half map (free), and the full map.

Figure 2.. Cryo-EM structure of rpsT P2-RPo.

(A) The Eco rpsT P2 promoter fragment used for cryo-EM. (B) rpsT P2-RPo cryo-EM density map (3.4 Å nominal resolution, low-pass filtered to the local resolution) is shown as a transparent surface and colored according to the key. The final model is superimposed. The DNA was modeled from −45 to +21. The t-strand DNA from −10 to −2, and the nt-strand DNA from −3 to +2 were disordered. (C) Top view of rpsT P2-RPo. DNA is shown as atomic spheres. Proteins are shown as molecular surfaces. Much of the β subunit is transparent to reveal the active site Mg²⁺ (yellow sphere), σ⁷⁰_finger, and DNA inside the RNAP active site cleft.

Figure 2—figure supplement 1.. rpsT P2-RPo cryo-EM processing pipeline.

Figure 2—figure supplement 2.. rpsT P2-RPo cryo-EM.

(A) The ten most populated classes from 2D classification. (B) Angular distribution for rpsT P2-RPo particle projections. (C) (top) The 3.4 Å resolution cryo-EM density map of rpsT P2-RPo is colored according to the key. The right view is a cross-section of the left view. (bottom) Same views as (top) but colored by local resolution (Cardone et al., 2013). (D) Gold-standard FSC of rpsT P2-RPo. The gold-standard FSC was calculated by comparing the two independently determined half-maps from RELION. The dotted line represents the 0.143 FSC cutoff, which indicates a nominal resolution of 3.4 Å. (E) FSC calculated between the refined structure and the half map used for refinement (work), the other half map (free), and the full map.

Figure 3.. Conformational flexibility of β'Si3 in TraR-Eσ⁷⁰.

(A) Overall view of TraR-Eσ⁷⁰ structure with alternative positions of Si3. Si3(I) is shown in brown. A ~ 121° rotation about the rotation axis shown gives rise to the position of Si3(II) shown in magenta. Si3 comprises two SBHM domains (Chlenov et al., 2005; Iyer et al., 2003), denoted SBHMa and SBHMb. The boxed region is magnified in (B). (B) Magnified view of TraR-Eσ⁷⁰(II) [same view as (A)]. The position of Si3(II) is outlined in magenta but the rest of Si3 is removed, revealing TraR behind. Three residues central to the TraR-Si3(II) interface (TraR-E46, R49, and K50) are colored yellow. (C) Orthogonal view as (B), showing the extensive TraR-Si3(II) interface. (D) – (G) Si3 interaction with TraR_G affects activation but not inhibition. Quantifications show averages with range from two independent experiments. (D) (top) Multi round in vitro transcription of rrnB P1 over a range of TraR concentrations (wedge indicates 2 nM - 2 µM) in the presence of WT-RNAP or ∆Si3-RNAP as indicated. Plasmid templates also contained the RNA-1 promoter. (bottom) Quantification of transcripts from experiments like those shown on (top) plotted relative to values in the absence of TraR. The IC₅₀ for inhibition by TraR was ~40 nM for both data sets. (E) (top) Multi round in vitro transcription of thrABC over a range of TraR concentrations (wedge indicates 2 nM - 2 µM) in the presence of 20 nM WT-RNAP or ∆Si3-RNAP as indicated. Plasmid templates also contained the RNA-1 promoter. (bottom) Quantification of transcripts from experiments like those shown on (top) plotted relative to values in the absence of TraR. (F) and (G) Multi round in vitro transcription of rrnB P1 (F) or pthrABC (G) was performed with 20 nM WT-Eσ⁷⁰ at a range of concentrations of WT or variant TraR (2 nM −2 µM). Transcripts were quantified and plotted relative to values in the absence of any factor (n = 2). For (F) IC₅₀ for inhibition by WT-TraR was ~50 nM, by E46A TraR was ~115 nM, R49A TraR was ~85 nM and by K50A TraR was ~30 nM.

Figure 3—figure supplement 1.. RNAP-Si3 interaction with TraR_G residues.

(A) - (G) Quantifications show averages with range from two independent experiments. (A) (B) (C) Multi round in vitro transcription of rpsT P2 (A), pargI (B) or phisG (C) was performed at a range of TraR concentrations (2 nM - 2 µM) in the presence of 20 nM WT- or ∆Si3-RNAP. Transcripts were quantified and plotted relative to values in the absence of TraR. Averages with range from two independent experiments are shown. (A) The IC₅₀ for inhibition by TraR was ~60 nM with WT-RNAP and ~90 nM with ∆Si3-RNAP. (D) (E) Multi round in vitro transcription from rrnB P1 (D) and pthrABC (E) was performed with 20 nM WT-Eσ⁷⁰ at a range of concentrations of WT- or variant TraR (2 nM - 2 µM). Transcripts were quantified and plotted relative to values in the absence of TraR. Error bars denote the standard deviation of three independent measurements. For (D), the IC₅₀ for inhibition by WT-TraR was ~50 nM, by P43A-TraR was ~80 nM, and by P45A-TraR was ~115 nM. (F) Effect of substitutions in TraR_G residues on binding to RNAP was determined by competition with ³²P-DksA in an Fe²⁺-mediated cleavage assay. WT-TraR (~0.6 µM), P43A-TraR (~3 μM), P45A-TraR (~4 μM), E46A-TraR (~1 μM), R49A-TraR (~1 μM) and K50A-TraR (~0.7 μM) reduced cleavage of 1.0 mM ³²P-DksA by 50%. Averages with range from two independent experiments are shown. (G) Transcription experiments were carried out with 20 nM WT- or ∆Si3-RNAP with 250 nM WT- or variant TraR as indicated. Values are relative to basal transcription by WT-RNAP without factor (normalized to1.0). Averages with range from two independent experiments are shown.

Figure 4.. TraR and the βlobe-Si1 domain.

(A) Overall top view of the TraR-Eσ⁷⁰ structure with the βlobe-Si1 in dark blue. The corresponding position of the βlobe-Si1 in the rpsT P2-RPo structure (Figure 2) is shown in light blue. The βlobe-Si1 of the rpsT P2-RPo structure (light blue) undergoes an ~18° rotation about the rotation axis shown to the βlobe-Si1 position in the TraR-Eσ⁷⁰ structure (dark blue), generating an extensive TraR-βlobe-Si1 interface. (B) Transcription of inhibited promoter rpsT P2 by 20 nM WT-RNAP or ΔSi1-RNAP with (+) or without (-) 250 nM TraR as indicated. Error bars denote standard deviation of three independent measurements. (C) Transcription of activated promoter pargI by 20 nM WT-RNAP or ΔSi1-RNAP with (+) or without (-) 250 nM TraR as indicated. Error bars denote standard deviation of three independent measurements. (D) (top) Multi-round in vitro transcription was carried out at a range of TraR concentrations (wedge indicates 4 nM - 4 μM) in the presence of 20 nM WT-Eσ⁷⁰ or EΔ1.1σ⁷⁰ as indicated. Plasmid template also contained the RNA-1 promoter. (bottom) Transcripts from experiments such as those in (top) were quantified and plotted relative to values in the absence of TraR with WT-Eσ⁷⁰ or EΔ1.1σ⁷⁰ with (+) or without (-) 250 nM TraR as indicated. Averages with range from two independent experiments are shown.

Figure 4—figure supplement 1.. EΔ1.1σ⁷⁰ has small defects for inhibition of rrnB P1 by TraR.

(A) (top) Multi round in vitro transcription at rrnB P1 was carried out at a range of TraR concentrations (wedge indicates 4 nM - 4 µM) in the presence of 20 nM WT-Eσ⁷⁰ or EΔ1.1σ⁷⁰ as indicated. Plasmid templates also contained the RNA-1 promoter. (bottom) Transcripts were quantified and plotted relative to values in the absence of TraR. The IC₅₀ for inhibition by TraR was ~50 nM with WT-RNAP and ~90 nM with Δ1.1σ⁷⁰-RNAP. Averages with range from two independent experiments are shown. (B) Basal level of transcription from rrnB P1 is only slightly affected by Δ1.1σ⁷⁰. Error bars denote standard deviation of three independent measurements. (C) Shown at the top is a proposed three-step linear kinetic scheme for RPo formation (Hubin et al., 2017a) with an added fourth irreversible step (formation of RP_itc) once RNA synthesis begins. The basal (WT-RNAP) free energy diagrams for hypothetical inhibited (top) and activated (bottom) promoters are shown in black (adapted from Galburt, 2018 as described in Materials and methods). The precise values for the input and output of the flux calculator are tabulated in Supplementary file 2. The proposed influence of deleting σ⁷⁰_1.1 on the energy diagram (lowering the kinetic barrier for the transition RP2 ⇄ RPo) is shown (green curves). The steady-state transcription output [calculated with the transcription flux calculator (Galburt, 2018) is represented by the circles on the right. The area inside the black circle represents the basal transcription output. Deleting σ⁷⁰_1.1 had no effect on transcription output from the inhibited promoter. The green circle (activated promoter) represents the effect of deleting σ⁷⁰_1.1 on the transcription output. Strong activation of a positively regulated promoter but little to no effect on a negatively regulated promoter correlates with our experimental results (Figure 4D, Figure 4—figure supplement 1A, B).

Figure 5.. TraR rotates the β'shelf and kinks the BH.

(A) Overall view if the TraR-Eσ⁷⁰(I) structure, shown as a molecular surface. The β'shelf domain is highlighted in hot pink. The β'shelf (which here includes the β'jaw) comprises Eco β' residues 787-931/1135-1150/1216-1317. The boxed region is magnified in (B). (B) Comparison of the rpsT P2-RPo BH-β'shelf (pink) and the TraR-Eσ⁷⁰ BH-β'shelf (hot pink). Binding of TraR induces an ~4.5° rotation (about the rotation axis shown) of the RPo-β'shelf to the position of the TraR-Eσ⁷⁰ β'shelf and a kink in the BH (circled region, which is magnified in (C)). (C) Focus on the region of the BH kink, which is centered near β'L788. The kink in the RPo BH is about 25°, while the kink in the TraR-Eσ⁷⁰ BH is about 29°. (D) View down the axis of the rpsT P2-RPo BH. The t-strand DNA, positioned at the RNAP active site (marked by the Mg²⁺ ion), closely approaches the BH. (E) View down the axis of the TraR-Eσ⁷⁰ BH. The BH kink induced by TraR binding sterically clashes with the position of the t-strand DNA (superimposed from the RPo structure).

Figure 6.. Multi-body analysis of Eσ⁷⁰ clamp conformational changes.

(A) Model of Eσ⁷⁰ refined into the consensus cryo-EM map (nominal 4.1 Å resolution). The RNAP clamp is highlighted in magenta. The clamp (which in the context of Eσ⁷⁰ includes σ⁷⁰₂) comprises the following Eco RNAP residues: β 1319–1342; β' 1–342, 1318–1344; σ⁷⁰92–137, 353–449. The mask used to analyze clamp motions by multi-body refinement (Nakane et al., 2018) is shown as a transparent yellow surface. (B - D) Histograms of Eigenvalue distributions (% of particles assigned each Eigenvalue from the dataset) for each of the three major principle components (Eigenvectors) from the multi-body analysis (Nakane et al., 2018). Each set of particles were divided into three equal-sized bins (red; gray; blue). The solid lines denote Gaussian fits to the histograms. (B) Component 1. (C) Component 2. (D) Component 3. (E - G) Three-dimensional reconstructions were calculated from the red and blue-binned particles for each principal component and models were generated by rigid body refinement. The models were superimposed using α-carbons of the RNAP structural core, revealing the alternate clamp positions shown (red and blue α-carbon ribbons with cylindrical helices). The σ⁷⁰_NCR, attached to the clamp but not included in the clamp motion analyses, is shown in faded colors. For each component, the clamp rotation and the direction of the rotation axis were determined (rotation axes are shown in gray). (E) Component 1 - clamp opening/closing. (F) Component 2 - clamp twisting. (G) Component 3 - clamp rolling.

Figure 7.. Range of clamp conformations for Eco RNAP complexes.

(top) Eσ⁷⁰ is shown as a molecular surface (α, ω, light gray; β, light cyan; β', light pink; σ⁷⁰, light orange) except the clamp/σ⁷⁰₂ module is shown schematically as blue or red outlines (the σ⁷⁰_NCR is omitted for clarity) to illustrate the direction and approximate range for the three major components of the clamp conformational changes (left, opening/closing; middle, twisting; left, rolling). (bottom) Histograms denote the range of clamp conformational changes for Eσ⁷⁰, TraR-Eσ⁷⁰, and rpsT P2-RPo, as indicated. The black bars denote the rotation range defined by dividing the Eigenvalue histograms into three equal bins and determining the clamp position for the red and blue bins (−33 %/+33% bin; see Figure 6). The gray bars denote the estimated rotation range to include 98% of the particles calculated from the Gaussian fits to the Eigenvalue histograms (1% of the particles excluded from each tail; see Figure 6).

Figure 8.. Proposed effects of TraR on the free energy diagram for hypothetical inhibited and activated promoters.

Shown at the top is a proposed three-step linear kinetic scheme for RPo formation (Hubin et al., 2017a) with an added fourth irreversible step (formation of RP_itc) once RNA synthesis begins. (T) denotes the presence of TraR, which must dissociate to allow the transition of RPo - > RPitc. The basal (WT-RNAP) free energy diagrams for hypothetical inhibited (top) and activated (bottom) promoters are shown in black (adapted from Galburt, 2018 as described in Materials and methods). The proposed influence of TraR on the energy diagram (lowering the kinetic barrier for the transition RP1 ⇄ RP2; lowering the free energy of RP2 relative to RPo; lowering the kinetic barrier for the transition RP2 ⇄ RPo) is shown (inhibited promoter, red curve; activated promoter, green curve) along with proposed links with the structural effects of TraR binding to RNAP described here. The steady-state transcription output [calculated with the transcription flux calculator (Galburt, 2018) is represented by the circles on the right. The precise values for the inputs and outputs for the flux calculator are tabulated in Supplementary file 2. The area inside the black circle represents the basal transcription output. The red or green circles (inhibited or activated promoters, respectively) represent the effect of TraR on the transcription output.