A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control


Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways, and the mechanisms that enable transit between them, remain largely unknown. Here, we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA molecules navigate cotranscriptional folding pathways.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Overview and in vitro characterization of the C. beijerinckii pfl riboswitch.
Fig. 2: C. beijerinckii pfl riboswitch folding intermediates.
Fig. 3: Mutagenesis of the C. beijerinckii pfl aptamer P3 stem.
Fig. 4: Mutagenesis of C. beijerinckii pfl aptamer pseudoknot and terminator base pairs.
Fig. 5: Classification of ZTP aptamer P3/L3 sequence and structure.
Fig. 6: A model for pfl riboswitch folding.

Data availability

Raw sequencing data that support the findings of this study have been deposited in the Sequencing Read Archive (http://www.ncbi.nlm.nih.gov/sra) with the BioProject accession code PRJNA510362. Individual BioSample accession codes are available in Supplementary Table 5. SHAPE-seq Reactivity Spectra generated in this work have been deposited in the RNA Mapping Database57 (http://rmdb.stanford.edu/repository/) with the accession codes ZTPRSW_BZCN_0001, ZTPRSW_BZCN_0002, ZTPRSW_BZCN_0003, ZTPRSW_BZCN_0004, ZTPRSW_BZCN_0005, ZTPRSW_BZCN_0006, ZTPRSW_BZCN_0007, ZTPRSW_BZCN_0008, ZTPRSW_BZCN_0009, ZTPRSW_BZCN_0010, ZTPRSW_BZCN_0011, ZTPRSW_BZCN_0012, ZTPRSW_BZCN_0013, ZTPRSW_BZCN_0014, ZTPRSW_BZCN_0015, ZTPRSW_BZCN_0016. Sample details are available in Supplementary Table 6. Source data for figures are available online and in the Northwestern University Arch Institutional Repository (https://doi.org/10.21985/N2220T). Uncropped gel images are shown in Supplementary Fig. 12. All other data that support the findings of this paper are available from the corresponding authors upon request.

Code availability

Spats v.1.0.1 can be accessed at https://github.com/LucksLab/spats/releases/. Scripts used in data processing are located at https://github.com/LucksLab/Cotrans_SHAPE-Seq_Tools/releases/ and https://github.com/LucksLab/LucksLab_Publications/tree/master/Strobel_ZTP_Riboswitch.


  1. 1.

    Pan, T. & Sosnick, T. RNA folding during transcription. Annu. Rev. Biophys. Biomol. Struct. 35, 161–175 (2006).

  2. 2.

    Zhang, J. & Landick, R. A two-way street: regulatory interplay between RNA polymerase and nascent RNA structure. Trends Biochem. Sci. 41, 293–310 (2016).

  3. 3.

    Lai, D., Proctor, J. R. & Meyer, I. M. On the importance of cotranscriptional RNA structure formation. RNA 19, 1461–1473 (2013).

  4. 4.

    Al-Hashimi, H. M. & Walter, N. G. RNA dynamics: it is about time. Curr. Opin. Struct. Biol. 18, 321–329 (2008).

  5. 5.

    Pan, T., Artsimovitch, I., Fang, X. W., Landick, R. & Sosnick, T. R. Folding of a large ribozyme during transcription and the effect of the elongation factor NusA. Proc. Natl Acad. Sci. USA 96, 9545–9550 (1999).

  6. 6.

    Heilman-Miller, S. L. & Woodson, S. A. Effect of transcription on folding of the Tetrahymena ribozyme. RNA 9, 722–733 (2003).

  7. 7.

    Wong, T. N., Sosnick, T. R. & Pan, T. Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures. Proc. Natl Acad. Sci. USA 104, 17995–18000 (2007).

  8. 8.

    Garst, A. D., Edwards, A. L. & Batey, R. T. Riboswitches: structures and mechanisms. Cold Spring Harb. Perspect. Biol. 3, a003533 (2011).

  9. 9.

    McCown, P. J., Corbino, K. A., Stav, S., Sherlock, M. E. & Breaker, R. R. Riboswitch diversity and distribution. RNA 23, 995–1011 (2017).

  10. 10.

    Nelson, J. W. & Breaker, R. R. The lost language of the RNA World. Sci. Signal. 10, eaam8812 (2017).

  11. 11.

    Howe, J. A. et al. Selective small-molecule inhibition of an RNA structural element. Nature 526, 672–677 (2015).

  12. 12.

    Kellenberger, C. A., Wilson, S. C., Sales-Lee, J. & Hammond, M. C. RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J. Am. Chem. Soc. 135, 4906–4909 (2013).

  13. 13.

    Porter, E. B., Polaski, J. T., Morck, M. M. & Batey, R. T. Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors. Nat. Chem. Biol. 13, 295–301 (2017).

  14. 14.

    Braselmann, E. et al. A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells. Nat. Chem. Biol. 14, 964–971 (2018).

  15. 15.

    Wickiser, J. K., Winkler, W. C., Breaker, R. R. & Crothers, D. M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 18, 49–60 (2005).

  16. 16.

    Frieda, K. L. & Block, S. M. Direct observation of cotranscriptional folding in an adenine riboswitch. Science 338, 397–400 (2012).

  17. 17.

    Perdrizet, G. A. II, Artsimovitch, I., Furman, R., Sosnick, T. R. & Pan, T. Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch. Proc. Natl Acad. Sci. USA 109, 3323–3328 (2012).

  18. 18.

    Watters, K. E., Strobel, E. J., Yu, A. M., Lis, J. T. & Lucks, J. B. Cotranscriptional folding of a riboswitch at nucleotide resolution. Nat. Struct. Mol. Biol. 23, 1124–1131 (2016).

  19. 19.

    Chauvier, A. et al. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation. Nat. Commun. 8, 13892 (2017).

  20. 20.

    Kim, P. B., Nelson, J. W. & Breaker, R. R. An ancient riboswitch class in bacteria regulates purine biosynthesis and one-carbon metabolism. Mol. Cell 57, 317–328 (2015).

  21. 21.

    Bochner, B. R. & Ames, B. N. ZTP (5-amino 4-imidazole carboxamide riboside 5′-triphosphate): a proposed alarmone for 10-formyl-tetrahydrofolate deficiency. Cell 29, 929–937 (1982).

  22. 22.

    Trausch, J. J., Marcano-Velazquez, J. G., Matyjasik, M. M. & Batey, R. T. Metal ion-mediated nucleobase recognition by the ZTP riboswitch. Chem. Biol. 22, 829–837 (2015).

  23. 23.

    Jones, C. P. & Ferre-D’Amare, A. R. Recognition of the bacterial alarmone ZMP through long-distance association of two RNA subdomains. Nat. Struct. Mol. Biol. 22, 679–685 (2015).

  24. 24.

    Ren, A., Rajashankar, K. R. & Patel, D. J. Global RNA fold and molecular recognition for a pfl riboswitch bound to ZMP, a master regulator of one-carbon metabolism. Structure 23, 1375–1381 (2015).

  25. 25.

    Schmidt, M. C. & Chamberlin, M. J. nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. J. Mol. Biol. 195, 809–818 (1987).

  26. 26.

    Rohlman, C. E. & Matthews, R. G. Role of purine biosynthetic intermediates in response to folate stress in Escherichia coli. J. Bacteriol. 172, 7200–7210 (1990).

  27. 27.

    Artsimovitch, I., Svetlov, V., Anthony, L., Burgess, R. R. & Landick, R. RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro. J. Bacteriol. 182, 6027–6035 (2000).

  28. 28.

    Artsimovitch, I. & Landick, R. Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. Proc. Natl Acad. Sci. USA 97, 7090–7095 (2000).

  29. 29.

    Widom, J. R. et al. Ligand modulates cross-coupling between riboswitch folding and transcriptional pausing. Mol. Cell 72, 541–552.e6 (2018).

  30. 30.

    Larson, M. H. et al. A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science 344, 1042–1047 (2014).

  31. 31.

    Vvedenskaya, I. O. et al. Interactions between RNA polymerase and the “core recognition element” counteract pausing. Science 344, 1285–1289 (2014).

  32. 32.

    Strobel, E. J., Yu, A. M. & Lucks, J. B. High-throughput determination of RNA structures. Nat. Rev. Genet. 19, 615–634 (2018).

  33. 33.

    Bevilacqua, P. C. & Assmann, S. M. Technique development for probing RNA structure in vivo and genome-wide. Cold Spring Harb. Perspect. Biol. 10, a032250 (2018).

  34. 34.

    Strobel, E. J., Watters, K. E., Nedialkov, Y., Artsimovitch, I. & Lucks, J. B. Distributed biotin–streptavidin transcription roadblocks for mapping cotranscriptional RNA folding. Nucleic Acids Res. 45, e109 (2017).

  35. 35.

    Komissarova, N. & Kashlev, M. Functional topography of nascent RNA in elongation intermediates of RNA polymerase. Proc. Natl Acad. Sci. USA 95, 14699–14704 (1998).

  36. 36.

    Reuter, J. S. & Mathews, D. H. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11, 129 (2010).

  37. 37.

    Sexton, A. N., Wang, P. Y., Rutenberg-Schoenberg, M. & Simon, M. D. Interpreting reverse transcriptase termination and mutation events for greater insight into the chemical probing of RNA. Biochemistry 56, 4713–4721 (2017).

  38. 38.

    Torgerson, C. D., Hiller, D. A., Stav, S. & Strobel, S. A. Gene regulation by a glycine riboswitch singlet uses a finely tuned energetic landscape for helical switching. RNA 24, 1813–1827 (2018).

  39. 39.

    Vvedenskaya, I. O. et al. Massively systematic transcript end readout, “MASTER”: transcription start site selection, transcriptional slippage, and transcript yields. Mol. Cell 60, 953–965 (2015).

  40. 40.

    Polaski, J. T., Kletzien, O. A., Drogalis, L. K. & Batey, R. T. A functional genetic screen reveals sequence preferences within a key tertiary interaction in cobalamin riboswitches required for ligand selectivity. Nucleic Acids Res. 46, 9094–9105 (2018).

  41. 41.

    Larson, M. H., Greenleaf, W. J., Landick, R. & Block, S. M. Applied force reveals mechanistic and energetic details of transcription termination. Cell 132, 971–982 (2008).

  42. 42.

    Hein, P. P. et al. RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement. Nat. Struct. Mol. Biol. 21, 794–802 (2014).

  43. 43.

    Geszvain, K. M. & Landick, R. in The Bacterial Chromosome (ed. Higgins, N. P.) Ch. 15 (American Society for Microbiology, 2005).

  44. 44.

    LeCuyer, K. A. & Crothers, D. M. Kinetics of an RNA conformational switch. Proc. Natl Acad. Sci. USA 91, 3373–3377 (1994).

  45. 45.

    LeCuyer, K. A. & Crothers, D. M. The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms. Biochemistry 32, 5301–5311 (1993).

  46. 46.

    Drogalis, L. K. & Batey, R. T. Requirements for efficient cotranscriptional regulatory switching in designed variants of the Bacillus subtilis pbuE adenine-responsive riboswitch. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/372573v1 (2018).

  47. 47.

    Zhao, B., Guffy, S. L., Williams, B. & Zhang, Q. An excited state underlies gene regulation of a transcriptional riboswitch. Nat. Chem. Biol. 13, 968–974 (2017).

  48. 48.

    Liberman, J. A. et al. Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics. Proc. Natl Acad. Sci. USA 112, E3485–E3494 (2015).

  49. 49.

    Savinov, A. & Block, S. M. Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element. Proc. Natl Acad. Sci. USA 115, 11976–11981 (2018).

  50. 50.

    Meyer, I. M. & Miklos, I. Co-transcriptional folding is encoded within RNA genes. BMC Mol. Biol. 5, 10 (2004).

  51. 51.

    Strobel, E. J. & Roberts, J. W. Two transcription pause elements underlie a σ70-dependent pause cycle. Proc. Natl Acad. Sci. USA 112, E4374–E4380 (2015).

  52. 52.

    Landick, R., Wang, D. & Chan, C. L. Quantitative analysis of transcriptional pausing by Escherichia coli RNA polymerase: his leader pause site as paradigm. Methods Enzymol. 274, 334–353 (1996).

  53. 53.

    Mortimer, S. A. & Weeks, K. M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129, 4144–4145 (2007).

  54. 54.

    Watters, K. E. et al. Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Methods 103, 34–48 (2016).

  55. 55.

    Pruitt, K. D., Tatusova, T. & Maglott, D. R. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 35, D61–D65 (2007).

  56. 56.

    Nawrocki, E. P. & Eddy, S. R. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29, 2933–2935 (2013).

  57. 57.

    Cordero, P., Lucks, J. B. & Das, R. An RNA mapping database for curating RNA structure mapping experiments. Bioinformatics 28, 3006–3008 (2012).

Download references


We thank R. Batey, C. Torgerson, S. Strobel, C. Jones and I. Artsimovitch for thoughtful discussions; J. Roberts (Cornell University) for providing E. coli NusA protein; J. Lis for use of facilities; J. Brink and S. Hockema for review of combinatorial mutagenesis alignment software; R. Breaker and K. Corbino for sharing a ZTP aptamer multiple sequence alignment; K. Watters for sharing a script to download RefSeq database entries. This work was supported by an Arnold O. Beckman Postdoctoral Fellowship (to E.J.S.), a New Innovator Award through the National Institute of General Medical Sciences of the National Institutes of Health (grant no. 1DP2GM110838 to J.B.L.), Searle Funds at The Chicago Community Trust (to J.B.L.) and by the National Institute of General Medical Sciences (grant no. T32GM008382). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

E.J.S. conceived the study and designed the experiments. E.J.S. and J.B.L. conceived the methodology. E.J.S. performed all RNA structure probing and combinatorial mutagenesis experiments. E.J.S., L.C. and K.E.B. performed targeted in vitro transcription experiments. P.D.C. performed molecular cloning. E.J.S. analyzed and interpreted data. E.J.S. wrote the software. E.J.S. and J.B.L. wrote the manuscript with input from L.C., K.E.B. and P.D.C.

Correspondence to Eric J. Strobel or Julius B. Lucks.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–12 and Supplementary Tables 1–6

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Strobel, E.J., Cheng, L., Berman, K.E. et al. A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control. Nat Chem Biol 15, 1067–1076 (2019). https://doi.org/10.1038/s41589-019-0382-7

Download citation

Further reading