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Structural basis of exoribonuclease-mediated mRNA transcription termination

Nature volume 628, pages 887–893 (2024)Cite this article

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Abstract

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1,2,3,4,5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5′-to-3′ exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5′ terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

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Fig. 1: The cryo-EM structure of Pol II Rat1–PTTC1.
Fig. 2: Protein interactions in Pol II Rat1–PTTC1.
Fig. 3: The RNA channel in Pol II Rat1–PTTC1.
Fig. 4: Rat1 rotates towards the RNA exit channel of Pol II as it shortens the nascent RNA.
Fig. 5: The proposed model of Rat1–Rai1-mediated Pol II termination.

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Data availability

Atomic coordinates have been deposited in the PDB under the accession codes 8JCH (Pol II Rat1–PTTC1) and 8K5P (Pol II Rat1–PTTC2). Corresponding cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-36162 (Pol II Rat1–PTTC1) and EMD-36908 (Pol II Rat1–PTTC2). Source data are provided with this paper.

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Acknowledgements

The work was supported by the National Key Research and Development Program of China 2018YFA0900701 (Y. Zhang) and the Basic Research Zone Program of Shanghai JCYJ-SHFY-2022-012 (Y. Zhang). We thank L. Kong, F. Wang, G. Li, J. Duan at electron microscopy system at the National Facility for Protein Science in Shanghai (NFPS), M. Zhang at the electron microscopy center in Interdisciplinary Research Center on Biology and Chemistry (IRCBC), and staffs X. Men, F. Liu, and S. Wang at electron microscopy center at Shuimu Biosciences in Hangzhou for providing technical support and assistance in cryo-EM data collection. We thank M. Zhang and Z. Zhang at the core facility of the Center for Excellence in Molecular Plant Sciences (CEMPS) for technical support and assistance in screening cryo-EM samples. The S. cerevisiae BJ2168 strain was a gift from G. Cai. The pESC-his vector was a gift from Z. Zhou.

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Authors and Affiliations

  1. Key Laboratory of Synthetic Biology, National Key Laboratory of Plant Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Yuan Zeng, Hong-Wei Zhang, Xiao-Xian Wu & Yu Zhang

  2. University of Chinese Academy of Sciences, Beijing, China

    Yuan Zeng & Hong-Wei Zhang

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  1. Yuan Zeng
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Contributions

Y. Zeng collected the cryo-EM data, solved the cryo-EM structures and carried out biochemical experiments. H.-W.Z. purified yeast Pol II. X.-X.W. assisted in structure determination. Y. Zhang designed experiments, analysed data and wrote the manuscript.

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Correspondence to Yu Zhang.

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Extended data figures and tables

Extended Data Fig. 1 The assembly of the Pol II Rat1-PTTC1 and Pol II Rat1-PTTC2.

a-b, Elution peaks and SDS-PAGE results of Pol II Rat1-PTTC1 from a size-exclusion chromatography. c-d, Elution peaks and SDS-PAGE result of Pol II Rat1-PTTC2 from a size-exclusion chromatography. Asterisks indicate degradation products of Rpb1. e, The Pol II release result shows the Rat1-Rai1 complex releases Pol II from the elongation complex. Three biological repeats were done, see Supplementary Fig.  1. f, The RNA cleavage result shows the Rat1-Rai1 complex has exoribonuclease activity. Three biological repeats were done, see Supplementary Fig.  1.

Source Data

Extended Data Fig. 2 Cryo-EM data processing of Pol II Rat1-PTTC1.

a, The flowchart of data processing for Pol II Rat1-PTTC1. b, Representative cryo-EM micrograph. c, Representative 2D class averages. d-f, Gold-standard FSC curves of map 1a, map 1b, and map 1c. g, The 3D FSC plot. The dotted line represents 0.143 cutoff of the global FSC curve. h, Nominal 2.7 Å-resolution cryo-EM reconstruction of map 2a filtered by local resolution. i-j, Map classified from Pol II Rat1-PTTC1. Map 1a represents the Pol II Rat1-PTTC1. Map 1c represents Pol II Spt4-Spt5 bound TEC. The Rat1-Rai1 map is colored green, and the Spt4-Spt5 map is colored blue.

Extended Data Fig. 3 Cryo-EM data processing of Pol II Rat1-PTTC2.

a, The flowchart of data processing for Pol II Rat1-PTTC2. b-c, Gold-standard FSC curves of map 2a, map 2b. d, The 3D FSC plot. The dotted line represents the 0.143 cutoff of the global FSC curve. e, Nominal 2.8 Å-resolution cryo-EM reconstruction of map 2a filtered by local resolution. f, Local refined map (map 2b) of Rat1, Rai1, Rpb4-Rpb7, and structure elements around the RNA exit channel of Pol II.

Extended Data Fig. 4 The interface between Rat1 and Rai1 in Pol II Rat1-PTTC1.

a, Overview of the interface between Rat1 and Rai1. The dashed boxes highlight the structure elements of Rat1 that contact the two concaves of Rai1. Green represents the Rat1 regions not observed in the crystal structure of S. pombe Rat1-Rai1 complex. b, Structural comparison of the Rat1-Rai1 complex in Pol II Rat1-PTTC1 and the crystal structure of S. pombe Rat1-Rai1 complex (PDB:3FQD26). c-d, The cryo-EM maps (map 1b) of interfaces 1 and 2.

Extended Data Fig. 5 The cryo-EM map of interfaces between Pol II and the Rat1-Rai1 complex in the structure of Pol II Rat1-PTTC1.

The view orientation and cartoon color are the same as in Fig. 2d. The cryo-EM map is colored in blue.

Extended Data Fig. 6 Sequence alignment of Rat1 homologs.

Sequence alignment shows that Rat1 residues in arms 1 and 2 contacting Pol II are generally conserved (red circles). Other Rat1 residues contacting Pol II are shown in blue circles.

Extended Data Fig. 7 Rat1 is incompatible with Pol II elongation factors.

a, Structural comparison between Pol II Rat1-PTTC1 and Pol II TEC (PDB:7XN755) shows the steric clash of Rat1 and Spt5 KOW1/2/3 domains. b, Structural comparison between Pol II Rat1-PTTC1 and Pol II TEC (PDB:7XN755) shows the steric clash of Rat1 and Spt6.

Extended Data Fig. 8 Rat1 can be recruited to Pol II independent of RNA.

a-b, Elution peaks and SDS-PAGE result of Pol II-RR from a size-exclusion chromatography without a nucleic-acid scaffold. Two biological repeats were done, see source data and Supplementary Fig.  1. c, The native-page result shows that the Rat1-Rai1(RR) complex slows the migration of Pol II in a concentration-dependent way, suggesting that the Rat1-Rai1 complex forms a stable complex with Pol II. Three biological repeats were done, see Supplementary Fig.  1. d, The native-page result shows that removing arms 1 and 2 weakens the interaction with Pol II. Three biological repeats were done, see Supplementary Fig.  1. e. The flag pull-down result shows Spt5 does not affect the interaction between Rat1-Rai1 and Pol II. Three biological repeats were done, see Supplementary Fig.  1.

Source Data

Extended Data Fig. 9 The interaction of nascent RNA with Rat1 in Pol II Rat1-PTTC1.

a, The interaction between the 5′ phosphate groups of the first two nucleotides with the active site of Rat1. Black dash, the coordination bonds. Blue dash, the H-bonds. b, The interaction of nascent RNA with Rat1 and Pol II. The cryo-EM map for the RNA-contacting residues is shown in blue. C, The modeled triphosphate group of the RNA 5′ terminus clashes with Rat1. Left, RNA with a monophosphate group in the structure of Pol II Rat1-PTTC1; right, RNA with a modeled triphosphate group in the structure of Pol II Rat1-PTTC1.

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

Supplementary Fig. 1

Uncropped gels from Extended Data Figs. 1 and 8.

Reporting Summary

Peer Review File

Source data

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Zeng, Y., Zhang, HW., Wu, XX. et al. Structural basis of exoribonuclease-mediated mRNA transcription termination. Nature 628, 887–893 (2024). https://doi.org/10.1038/s41586-024-07240-3

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