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Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand

Nature Structural & Molecular Biology volume 30, pages 902–913 (2023)Cite this article

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Abstract

Folding of nascent transcripts can be modulated by the RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (termed que-PEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination; however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-PEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, on ligand binding, the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

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Fig. 1: Mechanism of transcriptional pausing at the que pause and structure of the que-PEC.
Fig. 2: Transcription reactivation occurs through RNA reverse translocation in the presence of ligand.
Fig. 3: Binding of preQ1 to que-PEC RNAP induces swivel module rotation and β clamp closing.
Fig. 4: MDFF analysis reveals a rotation of the riboswitch within the RNA exit channel in the presence of ligand.
Fig. 5: Key RNA–protein interactions within the RNA exit channel.
Fig. 6: Model for RNAP entering and release from the que pause as a function of ligand binding to the riboswitch.

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Data availability

The cryo-EM volumes and maps have been deposited in the Electron Microscopy Data Bank (EMDB) and Protein Database (PDB), respectively. The accession numbers for the cryo-EM density maps reported in this paper are EMD-28845 (RNAP -preQ1 consensus), EMD-29640 (-preQ1 component 0), EMD-29676 (-preQ1 component 1), EMD-29683 (-preQ1 component 2), EMD-29732 (+preQ1 consensus), EMD-29812 (+preQ1 component 0) and EMD-29859 (+preQ1 component 1). The accession numbers for the atomic coordinates reported in this paper are PDB 8F3C (RNAP -preQ1 consensus), PDB 8G0O (-preQ1 component 0), PDB 8G1S(-preQ1 component 1), PDB 8G2W (-preQ1 component 2), PDB 8G4W (+preQ1 consensus), PDB 8GZE (+preQ1 component 0), PDB 8G8Z (+preQ1 component 1). Source data are provided with this paper.

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Acknowledgements

We thank J. Widom for the initial design of the que-PEC reconstitution, C. Scull for help with purification of RNAP mutant S1105A, I. Artsimovitch for helpful discussions, UM cryo-EM staff members and UM BSI and LSI for support of the UM cryo-EM facility. This work was supported by National Institutes of Health (NIH) R01 grants GM131922 and GM118524 to N.G.W. and NIH S10OD020011 and S10OD030275 to M.D.O. A portion of the molecular graphics and analyses was performed with UCSF Chimera and ChimeraX developed by the Resource for Biocomputing, Visualization and Informatics at UC San Francisco, with support from NIH P41-GM103311.

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Author notes

  1. Indrajit Deb

    Present address: Drug Discovery Unit, School of Life Sciences, University of Dundee, Dundee, UK

  2. These authors contributed equally: Adrien Chauvier, Jason C. Porta.

Authors and Affiliations

  1. Single Molecule Analysis Group and Center for RNA Biomedicine, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

    Adrien Chauvier, Emily Ellinger, Katarina Meze & Nils G. Walter

  2. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Jason C. Porta, Katarina Meze & Melanie D. Ohi

  3. Biophysics Program, University of Michigan, Ann Arbor, MI, USA

    Indrajit Deb & Aaron T. Frank

  4. Arrakis Therapeutics, Waltham, MA, USA

    Aaron T. Frank

  5. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Melanie D. Ohi

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Contributions

A.C., M.D.O. and N.G.W. conceived the project. A.C., J.C.P. and I.D. devised the methodology. A.C., J.C.P., I.D. and E.E. carried out the investigations. A.C. and J.C.P. wrote the original draft and A.C., J.C.P., I.D., E.E., K.M., M.D.O., A.T.F. and N.G.W. edited and reviewed the article. The work was supervised by A.T.F., M.D.O. and N.G.W. Funding was acquired by A.T.F., M.D.O. and N.G.W.

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Correspondence to Melanie D. Ohi or Nils G. Walter.

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Chauvier, A., Porta, J.C., Deb, I. et al. Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand. Nat Struct Mol Biol 30, 902–913 (2023). https://doi.org/10.1038/s41594-023-01002-x

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