title: Characterizing RNA Excited States Using NMR Relaxation Dispersion.
abstract: Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of noncoding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as "excited states." Compared to larger scale changes in RNA secondary structure, transitions toward excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around noncanonical motifs. Here, we describe a procedure for characterizing RNA excited states using off-resonance R1ρ NMR relaxation dispersion utilizing low-to-high spin-lock fields (25-3000 Hz). R1ρ NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data are then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited states. Application is illustrated with a focus on the transactivation response element from the human immune deficiency virus type 1, which exists in dynamic equilibrium with at least two distinct excited states.
authors.affiliation: Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xue
authors.affiliation: Department of Chemistry, Duke University, Durham, North Carolina, USA.
authors.forename: Dawn
authors.initials: D
authors.lastname: Kellogg
authors.affiliation: Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, USA.
authors.forename: Isaac J
authors.initials: IJ
authors.lastname: Kimsey
authors.affiliation: Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, USA.
authors.forename: Bharathwaj
authors.initials: B
authors.lastname: Sathyamoorthy
authors.affiliation: Biophysics Enhanced Program, University of Michigan Ann Arbor, Michigan, USA.
authors.forename: Zachary W
authors.initials: ZW
authors.lastname: Stein
authors.affiliation: Biophysics Enhanced Program, University of Michigan Ann Arbor, Michigan, USA.
authors.forename: Mitchell
authors.initials: M
authors.lastname: McBrairty
authors.affiliation: Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, USA; Department of Chemistry, Duke University, Durham, North Carolina, USA. Electronic address: hashim.al.hashimi@duke.edu.
authors.forename: Hashim M
authors.initials: HM
authors.lastname: Al-Hashimi
mesh_terms: D002244:Carbon / Q000737:chemistry*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D059370:RNA Folding; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D015533:Transcriptional Activation
chemical_list: D012367:RNA, Viral; D002244:Carbon; D009584:Nitrogen
keywords: Chemical exchange; HIV; NMR; RNA dynamics; RNA flexibility; RNA secondary structure; RNA switches; Relaxation dispersion; TAR