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Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70–RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

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Fig. 1: λPR promoter melting intermediates.
Fig. 2: Transcription bubble nucleation.
Fig. 3: Capture of T−7(nt) before A−11(nt).
Fig. 4: RNAP clamp closure is coupled to partial unfolding and ejection of σ701.1.
Fig. 5: σ701.1–H4 unfolding is necessary for RPo formation.
Fig. 6: Schematic overview of the initial steps of DNA opening at λPR after promoter binding.

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Data availability

Atomic coordinates used for data analysis (PDB 6PSQ, PDB 7MKD) are available from the Protein Data Bank (PDB; https://www.rcsb.org). All unique and stable reagents generated in this study are available without restriction from the lead contact, Seth A. Darst (darst@rockefeller.edu). The cryo-EM density maps and atomic coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and PDB as follows: RPc5°C (EMD-41456, PDB 8TOM), I1a (EMD-41433, PDB 8TO1), I1b (EMD-41439, PDB 8TO8), I1c (EMD-41448, PDB 8TOE) and I1d (EMD-41437, PDB 8TO6). Source data are provided with this paper.

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Acknowledgements

We thank E. A. Campbell, R. Gourse, D. Jensen, R. Landick, W. Ross and members of the Darst–Campbell laboratory for helpful discussions. We are grateful to the reviewers for their critiques, which improved the clarity and scientific rigor of this article. Some of the work reported here was conducted at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, supported by grants from the National Institutes of Health National Institute of General Medical Sciences (P41 GM103310), NYSTAR and the Simons Foundation (SF349247). A.U.M. received support through a fellowship from the Swiss National Science Foundation. A.U.M. is an Agouron Institute Awardee of the Life Sciences Research Foundation. This work was supported by the National Institutes of Health grant R35 GM118130 and Re-Entry Supplement R35 GM118130-04S1 to S.A.D.

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Author notes

  1. Brandon Malone

    Present address: Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute, New York, NY, USA

  2. James Chen

    Present address: Laboratory of Host-Pathogen Biology, The Rockefeller University, New York, NY, USA

  3. Venkata P. Dandey

    Present address: National Institute of Environmental Health Sciences, Durham, NC, USA

  4. Clinton S. Potter & Bridget Carragher

    Present address: Chan Zuckerberg Imaging Institute, San Francisco, CA, USA

  5. These authors contributed equally: Ruth M. Saecker, Andreas U. Mueller.

Authors and Affiliations

  1. Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA

    Ruth M. Saecker, Andreas U. Mueller, Brandon Malone, James Chen, Nina Molina & Seth A. Darst

  2. The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA

    William C. Budell, Venkata P. Dandey, Kashyap Maruthi, Joshua H. Mendez, Edward T. Eng, Laura Y. Yen, Clinton S. Potter & Bridget Carragher

  3. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Clinton S. Potter & Bridget Carragher

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R.M.S, A.U.M., J.C., B.M., C.S.P., B.C. and S.A.D. conceived the study. R.M.S, A.U.M., J.C., B.M. and N.M. performed protein purification and biochemistry analyses. R.M.S., A.U.M., J.C., B.M., W.C.B. and V.P.D. prepared cryo-EM specimens. R.M.S., A.U.M., J.C., B.M., V.P.D., K.M., J.H.M., E.T.E. and L.Y.Y. collected and processed cryo-EM data. R.M.S., A.U.M., B.M. and S.A.D. built the models and performed structural analysis. C.S.P., B.C. and S.A.D. acquired funding and supervised the project. R.M.S., A.U.M. and S.A.D. wrote the first draft of the manuscript; all authors contributed to finalizing the written manuscript.

Corresponding author

Correspondence to Seth A. Darst.

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The authors declare there are no competing interests.

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Nature Structural & Molecular Biology thanks Andreas Boland, Christian Dienemann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 λPR promoter fragment and tr-Spotiton.

a. λPR promoter DNA construct (−85 to +20) used for cryo-EM studies. The sequence from −40 to +10 is magnified below. b. Schematic diagram illustrating the principle of the tr-Spotiton device. For more details see ref. 21. c. Representative micrograph (dataset 4500 ms; t = 500 ms).

Extended Data Fig. 2 Cryo-EM processing pipeline for tr-Spotiton datasets.

Cryo-EM processing pipelines for Eco RNAP mixed with λPR DNA using tr-Spotiton. a. Datasets 1120 ms and 2120 ms (t = 120 ms, 8 mM CHAPSO). b. Dataset 3120 ms (t = 120 ms, 8 mM CHAPSO) c. Dataset 4500 ms (t = 500 ms, 8 mM CHAPSO).

Extended Data Fig. 3 Cryo-EM processing pipeline for combined datasets and comparison of particle population distributions.

a. Cryo-EM processing pipeline for combining polished particles from Spotiton datasets 1–3 (t = 120 ms) and dataset 4 (t = 500 ms; see Extended Data Fig. 2). b. Histogram plots showing the fraction of particles that contribute to each intermedate. The open bars show the mean particle fraction for the three 120 ms datasets ( < 123120 ms > ); the error bars denote ± SD for n = 3 (one point for each cryo-EM dataset). The gray bars denote the particle fraction for the single 500 ms dataset (4500 ms).

Source data

Extended Data Fig. 4 Cryo-EM of I1a, I1b, I1c, and I1d.

a.-d. Cryo-EM of I1a. a. Three views of the combined nominal 2.8 Å resolution cryo-EM map, filtered by local resolution77. The bottom row is colored according to the subunit (αI, αII, light grey; αCTD, limon; β, cyan; β‘, pink; σ70, orange; DNA t-strand, dark grey; DNA nt-strand, grey). The top row shows the same views but colored by local resolution77. The right view is a cross-section through the middle view. b. Directional 3D FSC, determined with 3DFSC90. c. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. d. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin. e.-h. Cryo-EM of I1b. e. Three views of the combined nominal 3.0 Å resolution cryo-EM map, filtered by local resolution77, colored as in (a). f. Directional 3D FSC, determined with 3DFSC90. g. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. h. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin. i.-l Cryo-EM of I1c. i. Three views of the combined nominal 3.0 Å resolution cryo-EM map, filtered by local resolution77, colored as in (a). j. Directional 3D FSC, determined with 3DFSC90. k. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. l. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin. m.-p Cryo-EM of I1d. m. Three views of the combined nominal 2.9 Å resolution cryo-EM map, filtered by local resolution77, colored as in (a). n. Directional 3D FSC, determined with 3DFSC90. o. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. p. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin.

Extended Data Fig. 5 Cryo-EM processing pipeline for dataset 5500 ms,FC8F.

Cryo-EM processing pipeline for Eco RNAP mixed with λPR DNA using tr-Spotiton (t = 500 ms, 1.5 mM FC8F).

Extended Data Fig. 6 Cryo-EM processing pipeline for 5 °C dataset.

a. λPR promoter DNA construct used for 5 °C cryo-EM studies. b. Cryo-EM processing pipeline for Eco RNAP and λPR DNA (−60 to +30) mixed manually and allowed to come to equilibrium at 5 °C (See Methods).

Extended Data Fig. 7 Cryo-EM of RPc5 °C, I1c5 °C, and I1d5 °C.

a.-d. Cryo-EM of RPc5 °C. a. Three views of the combined nominal 3.1 Å resolution cryo-EM map, filtered by local resolution77. The bottom row is colored according to the subunit (αI, αII, light grey; αCTD, limon; β, cyan; β‘, pink; σ70, orange; DNA t-strand, dark grey; DNA nt-strand, grey). The top row shows the same views but colored by local resolution77. The right view is a cross-section through the middle view. b. Directional 3D FSC, determined with 3DFSC90. c. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. d. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin. e.-h. CryoEM of I1c5 °C. e. Three views of the combined nominal 3.4 Å resolution cryo-EM map, filtered by local resolution77. The bottom row is colored according to the subunit (αI, αII, light grey; αCTD, limon; β, cyan; β‘, pink; σ70, orange; DNA t-strand, dark grey; DNA nt-strand, grey). The top row shows the same views but colored by local resolution77. The right view is a cross-section through the middle view. f. Directional 3D FSC, determined with 3DFSC90. g. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. h. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin. i.-l. CryoEM of I1d5 °C. i. Three views of the combined nominal 3.2 Å resolution cryo-EM map, filtered by local resolution77. The bottom row is colored according to the subunit (αI, αII, light grey; αCTD, limon; β, cyan; β‘, pink; σ70, orange; DNA t-strand, dark grey; DNA nt-strand, grey). The top row shows the same views but colored by local resolution77. The right view is a cross-section through the middle view. j. Directional 3D FSC, determined with 3DFSC90. k. Gold-standard FSC plot95, calculated by comparing two half maps. The dotted line represents the 0.143 FSC cutoff. l. Angular distribution plot, calculated in cryoSPARC. Scale depicts number of particles assigned to a specific angular bin.

Extended Data Fig. 8 Structural characteristics of λPR intermediates at 5 °C.

a. Comparison of λPR intermediates obtained by tr-Spotiton and at 5 °C. Eσ70 model of each state is shown as a transparent molecular surface, focusing on σ701.1 or duplex DNA occupying the RNAP cleft. Cryo-EM difference density (local-resolution filtered77) for σ701.1 (RPc5 °C) or for the DNA (I1d, RPo, I1d5 °C) is shown. The color-coding is shown in the legend at the upper left. The top row shows tr-Spotiton I1d and RPo (7MKD)8. The bottom row shows λPR-5 °C data (RPc5 °C and 1Id5 °C). A view of the entire structure (I1d5 °C) is shown in the lower right; the box denotes the region magnified in the other views. The orientation of the views shown is the same as the bottom view in each panel of Fig. 4. b. Each of the λPR-5 °C structures (RPc5 °C, I1c5 °C, I1d5 °C) were aligned pairwise with each of the tr-Spotiton structures (I1a, I1b, I1c, I1d) using the PyMOL align command on the RNAP structural core (α-carbons only), then the root-mean-square deviation was determined using the PyMOL rms_cur command over all matching α-carbons (the minimum number of overlapping α-carbon atoms was 3,709). The resulting rmsd values are plotted for each λPR-5 °C structure against each of the tr-Spotiton structures, showing that I1c5 °C matched most closely with I1c (orange arrow), and I1d5 °C matched most closely with I1d (red arrow).

Source data

Extended Data Fig. 9 The σ70 W-dyad and the −12 bp in λPR intermediates.

a. Top view of λPR-RPo (7MKD)8. Eσ70 is shown as a transparent molecular surface. The DNA is shown as atomic spheres, color-coded as in Fig. 2a. b-f. The boxed region in (a) is magnified, showing the region of the σ70 W-dyad and the −13 to −11 positions of the promoter. σ70 and DNA (color-coded as in Fig. 3) are shown in stick format; σ70 carbon atoms are colored orange but the W-dyad is highlighted in yellow. Transparent cryo-EM density (local-resolution filtered77) is superimposed. For reference, the positions of key RPo elements are shown in stick format and colored chartreuse (W-dyad in chair conformation and the −12 bp). For I1a, I1b, I1c, and I1d (b.-e.), the W-dyad is in the edge-on (wedge) conformation and the −12 bp is opened. Only in RPo is the W-dyad in the chair conformation and the −12 bp re-paired. b. I1a. c. I1b. d. I1c. e. I1d. f. RPo.

Extended Data Fig. 10 σ701.1 disulfide crosslinking, mass photometry, and a conformational change in the σ-finger.

a. The σ70 derivatives were analyzed by 10% SDS-polyacrylamide gel electrophoresis and visualized with Coomassie stain. Each σ70 derivative was analyzed under reducing (preventing formation of any disulfide bonds) or oxidizing (promoting the formation of disulfide bonds) conditions. Each Cys-pair mutant shows higher mobility under oxidizing conditions indicating formation of the relevant disulfide bond. Moreover, the difference in mobility between the reduced and oxidized condition correlates with the number of residues separating the two engineered Cys substitutions (I35C-S89C, 55 residues; Q8C-P32C, 25 residues; Y21C-Q54C, 34 residues). b. Average mass values and standard deviation determined by mass photometry of at least three individual reactions under oxidizing conditions of λPR promoter DNA (−60 to +30, see Extended Data Fig. 9a), σ70C-less, σ70I35C-S89C, core RNAP (E), and their respective holoenzyme complexes (E σ70C-less or E σ70I35C-S89C) with and without λPR are shown. The data are presented as mean values ± SD. A one-way analysis of variance (ANOVA) test was performed on all groups with a post-hoc analysis of planned comparisons using Fisher’s Least Significant Difference test with the p-values shown (only relevant comparisons are shown for visual clarity). c-j. Histograms of raw binding (positive mass)/unbinding (negative mass) event counts (binning of 5 kDa) where major individual peaks were approximated by a Gaussian distribution. Results of the curve fit for each replicate are displayed in the graphs. One raw frame from the recorded movie of one random replicate is shown above each graph. k. The λPR-RPo structure (7MKD)8 in the active-site region is shown; The RNAP is shown as a backbone cartoon (β, light cyan; β‘, light pink; σ70, orange); t-strand DNA is shown in stick format (carbon atoms dark grey); the RNAP active-site Mg2+ is shown as a yellow sphere. The structures of I1a, I1b, I1c, and I1d from the CHAPSO (light green) and FC8F (brown) datasets were superimposed by the RNAP structural core - shown is the σ-finger from each.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Supplementary Figs. 1 and 2.

Reporting Summary

Peer Review File

Supplementary Video 1

Supplementary Video 1. Slow motion videos (240 fps; iPhone 8S) of the grid robot in the Spotiton device after initiation of plunges at the motion settings shown. The total plunge time is the entire time the robot is in motion. The reported mixing times begin when the grid has passed the second (lower) dispenser, visible in the lower part of the video, and ends when it enters the liquid ethane (not seen). Plunges shown are for demonstration only. No grid is held in the tweezer.

Supplementary Video 2

Supplementary Video 2. 3DVA video 1 showing the RNAP clamp open/close mode (dataset 4500ms). At the beginning of the video, the RNAP clamp is open. The clamp (light green labels) and σ701.1 in the RNAP cleft (orange labels) are identified. As the clamp closes, σ701.1 disappears and density corresponding to downstream duplex DNA appears (yellow labels). The video continues through three additional cycles of opening and closing.

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Statistical Source Data

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Source Data Extended Data Fig. 10

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Saecker, R.M., Mueller, A.U., Malone, B. et al. Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01349-9

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