title: Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines.
abstract: Spliceosomes are multimegadalton RNA-protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition, and versatile structural dynamics. Single-molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues toward studying the mechanisms of biomolecular machines isolated directly from complex biological specimens, such as cell extracts. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy in exemplary single-molecule pull-down FRET studies of the yeast spliceosome and discuss the broad application potential of this technique.
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Kahlscheuer
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Widom
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA. Electronic address: nwalter@umich.edu.
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D005091:Exons; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D047468:Immunoprecipitation; D007438:Introns; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D012322:RNA Precursors; D012331:RNA, Fungal; C089187:cyanine dye 3; C085321:cyanine dye 5
keywords: Conformation; Pre-messenger RNA splicing; RNA dynamics; Single-molecule FRET; Spliceosome