Document Type
Post-Print
Publication Date
4-2010
Abstract
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.
DOI
10.1038/nsmb.1767
Publisher
Springer Nature
Repository Citation
Abelson, J., Blanco, M., Ditzler, M. A., Fuller, F., Aravamudhan, P., Wood, M., . . . Walter, N. G. (2010). Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nature Structural and Molecular Biology, 17(4), 504-512. https://doi.org/10.1038/nsmb.1767
Publication Information
Nature Structural and Molecular Biology