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

Autor:	Hua B; Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA., Jones CP; Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA., Mitra J; Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA., Murray PJ; T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, 21218, USA., Rosenthal R; T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, 21218, USA., Ferré-D'Amaré AR; Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA. adrian.ferre@nih.gov., Ha T; Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA. tjha@jhu.edu.; T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, 21218, USA. tjha@jhu.edu.; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA. tjha@jhu.edu.; Howard Hughes Medical Institute, Baltimore, MD, 21205, USA. tjha@jhu.edu.
Jazyk:	angličtina
Zdroj:	Nature communications [Nat Commun] 2020 Sep 10; Vol. 11 (1), pp. 4531. Date of Electronic Publication: 2020 Sep 10.
DOI:	10.1038/s41467-020-18283-1
Abstrakt:	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.
Databáze:	MEDLINE
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