Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Boyang Hua, Christopher P. Jones, Jaba Mitra, Peter J. Murray, Rebecca Rosenthal, Adrian R. Ferré-D’Amaré () and Taekjip Ha ()
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Boyang Hua: Johns Hopkins School of Medicine
Christopher P. Jones: Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health
Jaba Mitra: University of Illinois at Urbana-Champaign
Peter J. Murray: Johns Hopkins University
Rebecca Rosenthal: Johns Hopkins University
Adrian R. Ferré-D’Amaré: Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health
Taekjip Ha: Johns Hopkins School of Medicine

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: Abstract RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.

Date: 2020
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DOI: 10.1038/s41467-020-18283-1

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