Journal
NATURE STRUCTURAL & MOLECULAR BIOLOGY
Volume 17, Issue 4, Pages 504-U156Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nsmb.1767
Keywords
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Funding
- US National Institutes of Health (NIH) [GM062357, GM021119, GM077844]
- Rackham Merit Fellowship
- American Cancer Society
- Agouron Institute
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The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. During splicing in vitro, we observed a multitude of generally reversible time-and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3'-splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.
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