Title

Site-Directed Spin-Labeling Strategies And Electron Paramagnetic Resonance Spectroscopy For Large Riboswitches

Keywords

Continuous-wave-electron paramagnetic resonance; Electron paramagnetic resonance; Glycine riboswitch; Riboswitch; Site-directed spin labeling

Abstract

Genetic regulation effected by RNA riboswitches is governed by ligand-induced structural reorganization with modulation of RNA conformation and dynamics. Characterization of the conformational states of riboswitches in the presence or absence of salts and ligands is important for understanding how interconversion of riboswitch RNA folding states influences function. The methodology of site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is suitable for such studies, wherein site-specific incorporation of a nitroxide radical spin probe allows for local dynamics and conformational changes to be investigated. This chapter reviews a strategy for SDSL-EPR studies of large riboswitches and uses the full length 232 nucleotide (nt) kink-turn motif-containing Vibrio cholerae (VC) glycine riboswitch as an example. Spin-labeling strategies and the challenges of incorporating spin labels into large riboswitches are reviewed and the approach to overcome these challenges is described. Results are subsequently presented illustrating changes in dynamics within the labeled region of the VC glycine riboswitch as observed using SDSL-EPR.

Publication Date

1-1-2014

Publication Title

Methods in Enzymology

Volume

549

Issue

C

Number of Pages

287-311

Document Type

Article; Book Chapter

Personal Identifier

scopus

DOI Link

https://doi.org/10.1016/B978-0-12-801122-5.00013-1

Socpus ID

84925437371 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/84925437371

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