Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 17, Pages 6783-6788Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1219305110
Keywords
splicing mechanism; single-molecule FRET; RNA dynamics
Categories
Funding
- National Institutes of Health (NIH) [RO1s GM053007, GM43369, GM81648, GM759628]
- National Research Service Award Fellowship [GM079971]
- [K99/R00 GM086471]
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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.
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