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.
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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.
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.
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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.
The authors declare no competing interests.
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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
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