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.