title: Site-directed spin-labeling strategies and electron paramagnetic resonance spectroscopy for large riboswitches.
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.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida, USA.
authors.forename: Jackie M
authors.initials: JM
authors.lastname: Esquiaqui
authors.affiliation: Department of Chemistry, University of Central Florida, Orlando, Florida, USA.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Sherman
authors.affiliation: Department of Chemistry, University of Central Florida, Orlando, Florida, USA. Electronic address: yejingdong@gmail.com.
authors.forename: Jing-Dong
authors.initials: JD
authors.lastname: Ye
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida, USA. Electronic address: gefanucci@gmail.com.
authors.forename: Gail E
authors.initials: GE
authors.lastname: Fanucci
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005998:Glycine / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D058928:Riboswitch; D013113:Spin Labels / Q000138:chemical synthesis; D014734:Vibrio cholerae / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D058928:Riboswitch; D013113:Spin Labels; D005998:Glycine
keywords: Continuous-wave-electron paramagnetic resonance; Electron paramagnetic resonance; Glycine riboswitch; Riboswitch; Site-directed spin labeling