title: Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site.
abstract: Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Crawford
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Hoskins
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.forename: Jeff
authors.initials: J
authors.lastname: Gelles
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Moore
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D013057:Spectrum Analysis; D017381:Spliceosomes / Q000502:physiology*
chemical_list: D017931:DNA Primers; D009841:Oligonucleotides; D022821:RNA Splice Sites