title: Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics.
abstract: We report Single Molecule Cluster Analysis (SiMCAn), which utilizes hierarchical clustering of hidden Markov modeling-fitted single-molecule fluorescence resonance energy transfer (smFRET) trajectories to dissect the complex conformational dynamics of biomolecular machines. We used this method to study the conformational dynamics of a precursor mRNA during the splicing cycle as carried out by the spliceosome. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify the signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified an open conformation adopted late in splicing by a 3' splice-site mutant, invoking a mechanism for substrate proofreading. SiMCAn enables rapid interpretation of complex single-molecule behaviors and should prove useful for the comprehensive analysis of a plethora of dynamic cellular machines.
authors.affiliation: Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA.|Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Mario R
authors.initials: MR
authors.lastname: Blanco
authors.affiliation: Biology Department, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Martin
authors.affiliation: Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Kahlscheuer
authors.affiliation: Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Ramya
authors.initials: R
authors.lastname: Krishnan
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA.
authors.forename: John
authors.initials: J
authors.lastname: Abelson
authors.affiliation: Biology Department, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Alain
authors.initials: A
authors.lastname: Laederach
authors.affiliation: Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020134:Catalytic Domain; D016000:Cluster Analysis; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007438:Introns; D008390:Markov Chains; D009154:Mutation; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry*; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry; D017381:Spliceosomes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D000255:Adenosine Triphosphate