Abstract
Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription–translation-coupling quality control in Escherichia coli1,2,3,4. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho’s function.
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Data availability
Cryo-EM maps have been deposited into the EMDB with the following accession codes: EMD-27928, EMD-27929, EMD-27930, EMD-27931, EMD-27932, EMD-27933, EMD-27864, EMD-27865, EMD-27897, EMD-27913, EMD-27914, EMD-27915, EMD-27916, EMD-27917 and EMD-27918. Atomic coordinates have been deposited into the PDB with the following accession codes: 8E3F, 8E3H, 8E5K, 8E5L, 8E5O, 8E5P, 8E6W, 8E6X, 8E6Z and 8E70. Specific biological materials will be made available to qualified investigators on request.
References
Ray-Soni, A., Bellecourt, M. & Landick, R. Mechanisms of bacterial transcription termination. Annu. Rev. Biochem. 85, 319–347 (2016).
Mitra, P., Ghosh, G., Hafeezunnisa, M. & Sen, R. Rho protein: roles and mechanisms. Annu. Rev. Microbiol. 71, 687–709 (2017).
Roberts, J. Mechanisms of bacterial transcription termination. J. Mol. Biol. 431, 4030–4039 (2019).
Sunday, N., Svetlov, D. & Artsimovitch, I. in RNA Polymerases as Molecular Motors 2nd edn (eds Landick, R., Wang, J. & Strick, T) 100–131 (RSC Publishing, 2021).
Roberts, J. Termination factor for RNA synthesis. Nature 224, 1168–1174 (1969).
Bektesh, S. & Richardson, J. A ρ-recognition site on phage λ cro-gene mRNA. Nature 283, 102–104 (1980).
Brennan, C., Dombroski, A. & Platt, T. Transcription termination factor Rho is an RNA–DNA helicase. Cell 48, 945–952 (1987).
Dombroski, A. J. & Platt, T. Structure of Rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains. Proc. Natl Acad. Sci. USA 85, 2538–2542 (1988).
Alifano, P., Rivellini, F., Limauro, D., Bruni, C. & Carlomagno, M. A consensus motif common to all Rho-dependent prokaryotic transcription terminators. Cell 64, 553–563 (1991).
Jin, D. J., Burgess, R., Richardson, J. & Gross, C. Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and Rho. Proc. Natl Acad. Sci. USA 89, 1453–1457 (1992).
Sullivan, S. L. & Gottesman, M. E. Requirement for E. coli NusG protein in factor-dependent transcription termination. Cell 68, 989–994 (1992).
Seifried, S., Easton, J. & von Hippel, P. ATPase activity of transcription-termination factor rho. Proc. Natl Acad. Sci. USA 89, 10454–10458 (1992).
Geiselmann, J., Wang, Y., Seifried, S. & von Hippel, P. A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho. Proc. Natl Acad. Sci. USA 90, 7754–7758 (1993).
Steinmetz, E. & Platt, T. Evidence supporting a tethered tracking model for helicase activity of Escherichia coli Rho factor. Proc. Natl Acad. Sci. USA 91, 1401–1405 (1993).
Bogden, C., Fass, D., Bergman, N., Nichols, M. & Berger, J. The structural basis for terminator recognition by the Rho transcription termination factor. Mol. Cell 3, 487–493 (1999).
Skordalakes, E. & Berger, J. Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 114, 135–146 (2003).
Skordalakes, E. & Berger, J. Structural insights into RNA-dependent ring closure and ATPase activation by the Rho termination factor. Cell 127, 553–564 (2006).
Park, J. & Roberts, J. Role of DNA bubble rewinding in enzymatic transcription termination. Proc. Natl Acad. Sci. USA 103, 4870–4875 (2006).
Epshtein, V., Dutta, D., Wade, J. & Nudler, E. An allosteric mechanism of Rho-dependent transcription termination. Nature 463, 245–249 (2010).
Koslover, D., Fazal, F., Mooney, R., Landick, R. & Block, S. Binding and translocation of termination factor rho studied at the single-molecule level. J. Mol. Biol. 423, 664–676 (2012).
Lawson, M. R., Dyer, K. & Berger, J. M. Ligand-induced and small-molecule control of substrate loading in a hexameric helicase. Proc. Natl Acad. Sci. USA 113, 13714–13719 (2016).
Thomsen, N., Lawson, M., Witkowsky, L., Qu, S. & Berger, J. Molecular mechanisms of substrate-controlled ring dynamics and substepping in a nucleic acid-dependent hexameric motor. Proc. Natl Acad. Sci. USA 113, e7691–e7700 (2016).
Lawson, M. et al. Mechanism for the regulated control of bacterial transcription termination by a universal adaptor protein. Mol. Cell 71, 911–922 (2018).
Adhya, S. & Gottesman, M. Control of transcription termination. Annu. Rev. Biochem. 47, 967–996 (1978).
Richardson, J. P. Preventing the synthesis of unused transcripts by Rho factor. Cell 64, 1047–1049 (1991).
Burmann, B. et al. A NusE:NusG complex links transcription and translation. Science 328, 501–504 (2010).
Saxena, S. et al. Escherichia coli transcription factor NusG binds to 70S ribosomes. Mol. Microbiol. 108, 495–504 (2018).
Washburn, R. et al. Escherichia coli NusG links the lead ribosome with the transcription elongation complex. iScience 23, 101352 (2020).
Webster, M. & Weixlbaumer, A. Macromolecular assemblies supporting transcription–translation coupling. Transcription 12, 103–125 (2021).
Said, N. et al. Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase Rho. Science 371, eabd1673 (2021).
Hao, Z. et al. Pre-termination transcription complex: structure and function. Mol. Cell 81, 281–292 (2021).
Hao, Z., Svetlov, V. & Nudler, E. Rho-dependent transcription termination: a revisionist view. Transcription 12, 171–181 (2021).
Vassylyev, D., Vassylyeva, M., Perederina, A., Tahirov, T. & Artsimovitch, I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007).
Webster, M. et al. Structural basis of transcription–translation coupling and collision in bacteria. Science 369, 1355–1359 (2020).
Wang, C. et al. Structural basis of transcription–translation coupling. Science 369, 1359–1365 (2020).
Kang, J. et al. Structural basis for transcript elongation control by NusG family universal regulators. Cell 173, 1650–1662 (2018).
Boyer, P. The ATP synthase—a splendid molecular machine. Annu. Rev. Biochem. 66, 717–749 (1997).
Murayama, Y. et al. Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase. Preprint at bioRxiv https://doi.org/10.1101/2022.09.02.506315 (2022).
Guo, X. et al. Structural basis for NusA stabilized transcriptional pausing. Mol. Cell 69, 816–827 (2018).
Svetlov, V. & Artsimovitch, I. Purification of bacterial RNA polymerase: tools and protocols. Mol. Cell 26, 117–129 (2015).
Molodtsov, V. et al. Allosteric effector ppGpp potentiates the inhibition of transcript initiation by DksA. Mol. Cell 69, 828–839 (2018).
Artsimovitch, I. & Landick, R. Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. Proc. Natl Acad. Sci. USA 97, 7090–7709 (2000).
Sambrook, J., Fritsch, E. and Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, 1989).
Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).
Zheng, S. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–333 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Pettersen, E. et al. UCSF chimera—a visualization system for exploratory research and analysis. J. Comp. Chem. 25, 1605–1612 (2004).
Emsley, P., Lohkamp, B., Scott, W. & Cowtan, K. Features and development of Coot. Acta Cryst. D 66, 486–501 (2010).
Afonine, T., Headd, J., Terwilliger, T. and Adams, P. New tool: phenix.real_space_refine (Computatational Crystallography Newslettetter, 2013); https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2013_07.pdf.
Mastronarde, D. Advanced data acquisition from electron microscopes with SerialEM. Microsc. Microanal. 24, 864–865 (2018).
Acknowledgements
We thank J. Berger and I. Artsimovitch for plasmids and discussion; staff at the Rutgers CryoEM and Nanoimaging Facility, the National Center for CryoEM Access and Training (supported by National Institutes of Health (NIH) grant GM129539, Simons Foundation grant SF349247 and New York state grants), and the Stanford-SLAC Cryo-EM Center (supported by NIH grant GM129541) for microscope access; and staff at the IPS CMDH Center (supported by Shanghai Municipal Science and Technology Major Project 2019SHZDZX02) for computer resources. This work was supported by NIH grant GM041376 to R.H.E.
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V.M. and R.H.E. designed the experiments. V.M. prepared the proteins and nucleic acids and performed biochemical experiments. V.M., C.W., E.F. and J.T.K. performed cryo-EM data collection. V.M., C.W. and R.H.E. analysed the data. V.M., C.W. and R.H.E. prepared the figures. R.H.E. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Scaffold assay for Rho-dependent termination: additional data.
(a) Top, synthetic nucleic-acid scaffolds containing complementary nontemplate and template DNA strands (colours as in Fig. 1b). Bottom, RNA-release data assessing Rho-dependent termination in complexes containing NusG (left), NusG-N (center), or no NusG (right). Asterisks, released RNA products that exhibit increased levels relative to control reactions lacking Rho (lanes 6 and 7 of each subpanel). Termination is detected by an increase in released RNA products relative to control reactions lacking Rho. Assays were performed twice with consistent results. (b) As in (a), except that synthetic nucleic-acid scaffolds contain noncomplementary nontemplate and template DNA strands. The PBS ligands λtR1 rut and dC75 support efficient termination under these conditions (lanes 1 and 2 of each gel panel), but the shorter PBS ligands dC5 and dC15 do not (lanes 3 and 4 of each gel subpanel). It has been shown previously that dC15 and dC5 exhibit lower binding affinities for Rho than dC75, exhibiting half-maximally saturating concentrations 30-fold and 150-fold, respectively, the half-maximally saturating concentrations for dC7521. The PBS ligand λtR1 rut supports efficient and immediate termination under these conditions; with λtR1 rut, approximately 100 % of RNA is released before RNA extension (lane 1 of each gel panel). The PBS ligand dC75 supports efficient but less immediate termination under these conditions; with dC75, in the experiments of b, approximately 100% of RNA is released before RNA extension (lane 1 of each gel panel), but, in the experiments of a, approximately 30% of RNA is released before RNA extension, and approximately 70% of RNA is released only after RNA extension by 1 nt or 2 nt (lane 2 of each gel panel). The results indicate that both λtR1 rut and dC75 are effective PBS ligands and that λtR1 rut is a more effective PBS ligand than dC75 (see Extended Data Fig. 2).
Extended Data Fig. 2 Scaffold assay for Rho-dependent termination: additional data.
(a) Top, synthetic nucleic-acid scaffolds containing complementary nontemplate and template DNA strands (colours as in Fig. 1b; Extended Data Fig. 1). Bottom, RNA-extension data assessing Rho-dependent termination in complexes having λtR1 rut as PBS ligand (odd-numbered lanes) or dC75 as PBS ligand (even-numbered lanes) on scaffolds where the U-rich-RNA-segment length, n, is 4, 5, 6, 7, 8 or 9 codons (first, second, third, fourth, fifth, and sixth subpanels). Red asterisks, RNA products that exhibit increased levels compared to control reactions without PBS ligand; open circles, RNA products that exhibit decreased levels relative to control reactions without PBS ligand. Termination is detected by an increase in unextended and partly extended RNA products (lower and central bands), and a decrease in fully extended RNA products (top bands). Assays were performed twice with consistent results. (b) As in a, except that synthetic nucleic-acid scaffolds contain noncomplementary nontemplate and template DNA strands. The PBS ligand λtR1 rut supports efficient and immediate termination under these conditions; with λtR1 rut, approximately 100% of RNA synthesis ceases before RNA extension (lane 1 of each gel panel). The PBS ligand dC75 supports efficient but less immediate termination under these conditions; with dC75, approximately 30% of RNA synthesis ceases before RNA extension, and approximately 70% of RNA synthesis ceases only after RNA extension by 1 nt or 2 nt (lane 2 of each gel panel). The results indicate that both λtR1 rut and dC75 are effective PBS ligands and that λtR1 rut is a more effective PBS ligand than dC75 (see Extended Data Fig. 1). All six tested U-rich-RNA-segment lengths (n = 4, 5, 6, 7, 8, and 9 codons) support termination. In the experiments of a, the six tested U-rich-RNA-segment lengths support termination comparably efficiently. In the experiments of b, shorter lengths (n = 4, 5, and 6 codons) support termination more efficiently than longer lengths (n = 7, 8, and 9 codons).
Extended Data Fig. 3 Structure determination: λtR1-NusG-Rho-TEC (n = 6).
(a) Data processing scheme (Table S1). (b) Representative electron microphotograph and 2D class averages (50 nm scale bar in right subpanel). (c) Orientation distribution. (d) Fourier-shell correlation (FSC) plot. (e) EM density map coloured by local resolution (view orientation as in Fig. 2a, left). (f-i) Representative EM density (blue mesh) and fits (ribbons) for RNAP regions that interact with Rho, NusG, Rho protomer C, PBS ligand interacting with Rho protomer C, spacer RNA, and SBS ligand.
Extended Data Fig. 4 Structure determination: dC75-NusG-Rho-TEC (n = 6).
(a) Data processing scheme (Table S1). (b) Representative electron microphotograph and 2D class averages (50 nm scale bar in right subpanel). (c) Orientation distribution. (d) Fourier-shell correlation (FSC) plot. (e) EM density map coloured by local resolution (view orientation as in Fig. 2a, left). (f-i) Representative EM density (blue mesh) and fits (ribbons) for RNAP regions that interact with Rho, NusG, Rho protomer C, PBS ligand interacting with Rho protomer C, spacer RNA, and SBS ligand.
Extended Data Fig. 5 Structure determination: NusG-Rho-TEC (n = 6, 7, and 8).
(a) Data processing scheme (n = 8). (b) Representative electron microphotograph and 2D class averages (n = 8; 50 nm scale bar in right subpanel). (c) Orientation distribution (n = 8). (d) Fourier-shell correlation (FSC) plot. (e) EM density map coloured by local resolution (n = 8) (view orientation as in Fig. 2a, left). (f) EM density maps for NusG-Rho-TEC obtained using nucleic acid scaffolds with n = 6, 7, and 8 (view orientation as in Fig. 2a, left). (g-j) Representative EM density (blue mesh) and fits (ribbons) for RNAP regions that interact with Rho, NusG, Rho protomer C, spacer RNA, and SBS ligand.
Extended Data Fig. 6 Comparison of structure of λtR1-NusG-Rho-TEC to structures of Rho-TEC complexes of 30 and 31.
(a) Structure of λtR1-NusG-Rho-TEC. Rho N-terminal domain (Rho-N); cyan, and Rho C-terminal domain (Rho-C), slate blue, are shown in different colours to highlight orientation of Rho domains relative to the TEC. View orientation and other colours as in Fig. 2a, left. (b)-(c) Structures of Rho-TEC complexes of 30 (B; PDB 6Z9R) and 31 (C; PDB 6XAS; NusA omitted for clarity). View orientation that superimposes TEC atoms in b and c on TEC atoms in a. Colours as in a. The ring opening of the open-ring Rho hexamer in structures of 30 and 31 is indicated by a dashed line, and rotations and translation that relate the orientation of Rho relative to the TEC in structures of 30 and 31 to those in a are summarized below.
Extended Data Fig. 7 Protein-RNA interactions between PBS ligand and Rho PBS.
(a) Rho-(PBS ligand) interactions in dC75-NusG-Rho-TEC. View orientations and colours as in Fig. 3a, top right. (b) Rho-(PBS ligand) interactions in crystal structure of Rho hexamer interacting with six copies of oligoribonucleotide in absence of NusG and TEC (PDB 2HT1)17. View orientations and colours as in Fig. 3a, top. Oligoribonucleotides non-specifically interacting with Rho-C omitted for clarity. (c)-(d) Rho-(PBS ligand) interactions in structures of 30 (c; PDB 6Z9R) and 31 (d; PDB 6XAS). Dashed black lines, ring openings in open-ring Rho hexamers in structures of 30 and 31; black cylinders and black ribbons, RNAP or DNA structural elements of that occlude PBS-binding sites, preventing interaction with PBS ligand in structures of 30 and 31. View orientations and other colours as in Fig. 3a, top.
Extended Data Fig. 8 Protein-protein interactions between NusG-C and Rho.
(a) Rho-(NusG-C) interactions in λtR1-NusG-Rho-TEC. Colours as in Extended Data Fig. 6. (b) Superimposition of NusG-C and Rho protomer C of λtR1-NusG-Rho-TEC (coloured as in a) on structure of one NusG-C and one Rho protomer in crystal structure of Rho hexamer interacting with six copies of a NusG-C protein fragment in absence of TEC (PDB 6DUQ; grey)23. Dashed grey ribbon, disordered segment of NusG-C β8-β9 loop in crystal structure. The interaction between NusG-C and Rho protomer B (see a) is not present in the crystal structure of Rho hexamer interacting with six copies of NusG-C protein fragment23 (see b), apparently because steric clash between adjacent NusG-C protein fragments in that structure resulted in disorder of the NusG β8-β9 loop and disruption of interactions made by the NusG β8-β9 and β10-β11 loops (dashed lines in b).
Extended Data Fig. 9 Motor states of Rho hexamer.
(a) Left, superimposition of structure of λtR1-NusG-Rho-TEC (coloured as in Fig. 2a, left) on crystal structure of Rho hexamer interacting with SBS ligand and Mg-ADP-BeF3 in absence of NusG and TEC (PDB 5JJI; coloured grey)22. View orientation as in Fig. 2a, left. TEC omitted for clarity. Right, superimposition of Rho protomers A-F interacting with PBS ligand, SBS ligand, and Mg-ADP-BeF3 in structure of λtR1-NusG-Rho-TEC (coloured as in a) on crystal structure of Rho protomers A-F interacting with SBS ligand and Mg-ADP-BeF3 (PDB 5JJI; grey)22. (b) Occupancy and order of ATP-binding sites of λtR1-NusG-Rho-TEC. Figure presents EM density (blue mesh) and fit (cyan for Rho; orange, light orange, and yellow for Mg-ADP-BeF3 at high, low, and very low occupancies, respectively) for ATP binding sites between Rho protomers A and B, B and C, C and D, D and E, E and F, and F and A.
Extended Data Fig. 10 Comparison of structure of λtR1-NusG-Rho-TEC to structures of functional transcription-translation complexes NusG-TTC-B and NusA-NusG-TTC-B.
(a) Structure of λtR1-NusG-Rho-TEC. View orientation and colours as in Fig. 2a, left. (b)-(c) Structures of NusG-TTC-B (B; PDB 6XII)34,35 and NusA-NusG-TTC-B (C; PDB 6X7F)35. View orientation that superimposes TEC atoms in b and c on TEC atoms in a. Ribosome 30S subunit, yellow; ribosome 50S subunit, grey; P- and E-site tRNAs bound to ribosome, green and orange; NusA, light blue. Other colours as in a.
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This file contains Supplementary Fig. 1 (unprocessed gel images) and Supplementary Table 1 (summary of cryo-EM data collection and data processing).
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Molodtsov, V., Wang, C., Firlar, E. et al. Structural basis of Rho-dependent transcription termination. Nature 614, 367–374 (2023). https://doi.org/10.1038/s41586-022-05658-1
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DOI: https://doi.org/10.1038/s41586-022-05658-1
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