期刊
METHODS
卷 125, 期 -, 页码 45-54出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2017.05.011
关键词
Single-molecule; FRET; Fluorescence; Spliceosome; RNA; Dynamics
资金
- National Institutes of Health [R01 GM112735]
- Shaw Scientist and Beckman Young Investigator Awards
- University of Wisconsin-Madison
- Wisconsin Alumni Research Foundation (WARF)
- Department of Biochemistry
The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub populations of molecules with unique behaviors. When smFRET is combined with single-molecule fluorescence colocalization, conformational dynamics can be further linked to the presence or absence of a given spliceosome component. Here, we provide a description of experimental considerations, approaches, and workflows for smFRET with an emphasis on applications for the splicing machinery. (C) 2017 Elsevier Inc. All rights reserved.
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