title: 2-Aminopurine Fluorescence as a Probe of Local RNA Structure and Dynamics and Global Folding.
abstract: The biology of an RNA is encoded in its structure and dynamics, whether that be binding to a protein, binding to another RNA, enzymatic catalysis, or becoming a substrate. In solution, most RNA molecules are sampling conformations, and their structures are best described as conformational ensembles. For larger RNAs, experiments that can describe the conformations of their domains can be particularly daunting, especially when the RNA is novel and not well characterized. Here, we explain how we have used site-specific 2-aminopurine as a fluorescent probe of the secondary and tertiary structures of a 60 nucleotide RNA, and what new findings we have about its Mg(2+)-dependent conformational changes. We focus on this RNA from prokaryotic ribosome as a proof of concept as well as a research project. Its tertiary structure is known from a cocrystal, and its secondary structure is modeled from phylogenetic conservation, but there are virtually no data describing the motions of its nucleotides in solution, or its folding kinetics. It is a perfect system to illustrate the unique information that comes from a comprehensive fluorescence study of this intricate RNA.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, Missouri, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rau
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, Missouri, USA. Electronic address: kathleenhal@gmail.com.
authors.forename: Kathleen B
authors.initials: KB
authors.lastname: Hall
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry*; D001483:Base Sequence; D002413:Cations, Divalent; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000648:ultrastructure*; D005453:Fluorescence; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D059370:RNA Folding; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000648:ultrastructure*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000648:ultrastructure*; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D015335:Molecular Probes; D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins; C525862:rplK protein, E coli; D015075:2-Aminopurine; D008274:Magnesium
keywords: Mg(2+)-dependent RNA folding; RNA folding kinetics; Steady-state 2AP fluorescence; Stopped-flow fluorescence; Time-resolved 2AP fluorescence