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Structural basis for intrinsic transcription termination

Abstract

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4—called intrinsic termination transcription in bacteria—in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

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Fig. 1: The TTC-pause complex.
Fig. 2: The TTC-hairpin complex.
Fig. 3: The release complex of transcription termination.
Fig. 4: The proposed pathway of intrinsic termination.

Data availability

The cryo-EM map and coordinates have deposited in Protein Data Bank with accession numbers 7YP9 (TTC pause), 7YPA (TTC hairpin) and 7YPB (TTC release) and in the Electron Microscopy Data Bank under accession numbers EMD-33996 (TTC pause), EMD-33997 (TTC hairpin) and EMD-33998 (TTC release). Source data are provided with this paper.

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Acknowledgements

The work was supported by Strategic Priority Research Program of the CAS XDB29020000 (Y. Zhang), National Key Research and Development Program of China 2018YFA0903701 (Y. Zhang), Basic Research Zone Program of Shanghai JCYJ-SHFY-2022-012 (Y. Zhang), Shanghai Science and Technology Innovation program (19JC1415900) (Y. Zhang), and US National Institute of General Medical Sciences, NIH (GM38660) (R.L.). We thank L. Kong, F. Wang, G. Li and J. Duan at the Cryo-EM Center of NFPS in Shanghai and S. Chang at the Cryo-EM Center of Zhejiang University for assistance in cryo-EM data collection.

Author information

Authors and Affiliations

Authors

Contributions

L.Y. collected the cryo-EM data and solved the cryo-EM structures. L.Y., E.O.O., C.Y. and R.A.M. designed, performed and analysed biochemical experiments. J.S., L.S., X.W., A.W., D.H., Y. Zeng and Y.F. assisted in structure determination. R.L. and Y. Zhang designed experiments, analysed data and wrote the manuscript.

Corresponding authors

Correspondence to Yu Feng, Robert Landick or Yu Zhang.

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The authors declare no competing interests.

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Nature thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 The processing pipeline for single-particle reconstitution of E. coli TTC-pause.

a. Elution peaks of TTC pause (peak1) from a size-exclusion column. b. The SDS-PAGE and c. the native-PAGE of peak 1. The gel was first stained with SYBR Gold for nucleic acids and then with Coomassie Brilliant Blue for proteins. The experiment has been repeated three times with similar results. d. The flowchart of data processing of TTC pause. e. The 3D FSC plot. The dotted line represents 0.143 cutoff of the global FSC curve. f. The angular distribution of single-particle projections by number of particles of each projection. g. The cryo-EM map of TTC pause colored by local resolution. h. The cryo-EM map of TTC pause in front and side views.

Source Data

Extended Data Fig. 2 The detailed analysis of TTC-pause structure.

a. The previously determined consensus sequence for elemental pause sites18. b. The consensus sequence features from 100 previously identified E. coli intrinsic terminators11. c. The structural comparison of downstream dsDNA between TTC pause (pink and red) and TEC (gray). d. The structural comparison of RNA-DNA hybrid between TTC pause (pink and red) and TEC (gray). e. The path of single-stranded ntDNA of the transcription bubble in RNAP. f. The detailed interactions between single-stranded ntDNA of the transcription bubble and RNAP. Blue mesh, the cryo-EM map. Black dash, H-bond.

Extended Data Fig. 3 Disruption of NT –4 pocket interaction does not affect termination efficiency or pre-termination pausing.

a. NT –4 pocket with R180, R465 and V469 shown as sticks. An adenine on the non-template DNA is flipped into the pocket and hydrogen bonds with R465. b. DNA template encoding λPR promoter and λtR2 terminator used for promoter-initiated, in vitro transcription assay of the effects of pocket mutants on termination. c. Effect of pocket mutants on the termination efficiency of wild-type λtR2 terminator. d. Effect of pocket mutants on the termination efficiency of a λtR2 terminator containing a different downstream DNA. e. Effect of pocket mutants on the termination efficiency of ϕ82t500 terminator. f. Effect of pocket mutants on the termination efficiency of a scaffold-based λtR2 terminator in the absence or presence of E. coli NusA. Data are presented as mean ± SD, n = 3 biologically independent experiments for c-f. g. Effect of pocket mutants on pre-termination pausing at U7 and U8. A variant of λtR2 terminator lacking the upstream half of the terminator hairpin (pRM1234; Supplementary Table 1) and an antisense DNA to prevent backtracking of G33 complexes were used. See legend to Extended Data Fig. 8c and methods for more details. Results from a single replicate are shown. Raw data for the gels in this figure can be found in Supplementary Fig. 1a–d.

Source Data

Extended Data Fig. 4 The complex assembly and processing pipeline for cryo-EM map construction of TTC hairpin.

a. The flowchart of data processing of TTC hairpin. b. The 3D FSC plot. The dotted line represents 0.143 cutoff of the global FSC curve. c. The angular distribution of single-particle projections by number of particles of each projection. d. The cryo-EM map colored by local resolution. (e) The cryo-EM map of TTC hairpin in front and side views.

Extended Data Fig. 5 The detailed analysis of TTC-hairpin structure.

a. The comparison of nuclei-acid scaffolds in TTC hairpin (red) and TTC pause (gray). b. A clear path across the cleft between β CTR and β′ ZBD for the 5′-end ssRNA that leads into the bottom of the RNA exit channel. The insert shows a positively charged groove that likely guides 5′-proximal ssRNA upstream of the hairpin stem out of the RNA exit channel. The modeled 5′-proximal ssRNA is shown in green. The electrostatic potential surface of RNAP was generated using APBS tools in Pymol. c. Structural comparison of the upstream RNA-DNA hybrids in TTC hairpin (blue and pink) and TEC (gray; PDB: 6ALF). The two structures were superimposed based on the RNAP-β′ rudder and lid motifs. d. The schematic presentation of the register of the RNA-DNA hybrid in TTC hairpin and a TEC in a pre-translocation state. e. G(–10) of the tDNA (cyan) is modeled to flip from its previous position (gray) through the tunnel to pair with the C(–10) of the ntDNA. f. The cryo-EM map shows that the −10 base pair has the weakest signal in the RNA-DNA hybrid.

Extended Data Fig. 6 The complex assembly and processing pipeline for cryo-EM map construction of TTC release.

a. Elution peaks of TTC pause (peak1) from a size-exclusion column. b. The SDS-PAGE of TTC-pause. c. The native-PAGE of TTC pause and TTC release. The gel was visualized by Cy5-fluorescein labeled at the 5′ terminus of the nascent RNA. d. The flowchart of data processing of TTC release. e. The 3D FSC plot. The dotted line represents 0.143 cutoff of the global FSC curve. f. The angular distribution of single-particle projections by number of particles of each projection of map A. g. The map A colored by local resolution. h. The cryo-EM map of TTC release in front and side views.

Source Data

Extended Data Fig. 7 The detailed analysis of the TTC-release structure.

a. The comparison of upstream dsDNA in TTC release (pink and red) and TTC hairpin (gray). The last base pair of the upstream DNA in TTC release and TTC hairpin was colored as blue and green, respectively. The dashes indicate helical axis of the upstream DNA of the two structures. b. The modeled upstream base pairs (–17 to –12; gray) show loss of contact with RNAP and explain loss of cryo-EM map signals in TTC release. The map and model of the upstream DNA in TTC release are shown in red and pink. c. The comparison of downstream dsDNA in TTC release (pink and red) and TEC (gray; PDB: 6ALF). d. The comparison of main cleft in TTC release (brown) and TEC (green).

Extended Data Fig. 8 Rewinding of transcription bubble triggers RNA release.

a. The sequence of DNA templates used in Fig. 3g. b. The in vitro transcription assay suggests that dsDNA rewinding is required for intrinsic termination at the ϕ82t500 terminator. Data are presented as mean ± SD, n = 3 biologically independent experiments, ****P < 0.0001; two-tailed unpair t-tests. Raw data for the gels can be found in Supplementary Fig. 1g. c. Mapping the hairpin stem distance from U8 that triggers termination at U7 or U8. A template encoding a variant of λtR2 terminator lacking the upstream half of the terminator hairpin was used (from pRM1234, Supplementary Table 1). Core-RNAP was halted at G33, and backtracking was prevented by addition of asDNA (#14483). Transcription was restarted after adding different asRNAs and a master mix with (ATP + GTP + CTP to yield final concentrations of 150 μM, and UTP to 10 μM). Samples were collected and mixed with stop buffer after 10, 20, 30, and 60 sec. The remaining fraction was then chased by all 4 NTPs to a final concentration of 650 μM each. Termination starts taking place when the hairpin is −11 away from U8. Results from a single replicate are shown. Raw data for the gel can be found in Supplementary Fig. 1h.

Source Data

Extended Data Fig. 9 The proposed pathways of hairpin extension after the TTC -hairpin intermediate.

The choice between the alternative pathways of hybrid-shearing and hypertranscloation depends on terminator sequences26. It is also possible that hairpin completion follows a third alternative pathway, ‘hairpin invasion’12,25,28,36, in which the RNAP clamp opens to allow hairpin extension into the main cleft. Although clamp opening cannot be obligatory for RNA release30, the RNAP clamp is in constant thermal motion52,53, can open fully in some conditions36,37,38,39, and thus in principle could open upon DNA rewinding and allow the hairpin stem to extend into the main cleft without steric hinderance. After hairpin extension aided by bubble collapse, RNA release leads to TTC release (see Fig. 4).

Extended Data Table 1 The statistics of cryo-EM structures in this study

Supplementary information

Supplementary Information

A list of oligonucleotides and plasmids (Supplementary Table 1) and uncropped blots (Supplementary Fig. 1).

Reporting Summary

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Supplementary Video 1

Cryo-EM map and structural analysis of TTC pause.

Supplementary Video 2

Cryo-EM map and structural analysis of TTC hairpin.

Supplementary Video 3

A proposed route for rewinding of the −10 nucleotides.

Supplementary Video 4

Cryo-EM map and structural analysis of TTC release.

Source data

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You, L., Omollo, E.O., Yu, C. et al. Structural basis for intrinsic transcription termination. Nature 613, 783–789 (2023). https://doi.org/10.1038/s41586-022-05604-1

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