Pulsed EPR Methods in the Angstrom to Nanometre Scale Shed Light on the Conformational Flexibility of a Fluoride Riboswitch

20 August 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F- cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the MgII-bound apo, and the F--bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the F--bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing longer distances, revealed varying conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.

Keywords

ENDOR
PELDOR
DEER
fluorine
RNA

Supplementary materials

Title
Description
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Title
ComparativeDEERAnalyzer
Description
Output of the ComparativeDEERAnalyzer
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Supplementary Information
Description
Supplementary materials, methods and results
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