Preparation of fluorescent pre-mRNA substrates for an smFRET study of pre-mRNA splicing in yeast.
Methods Enzymol
; 472: 31-40, 2010.
Article
em En
| MEDLINE
| ID: mdl-20580958
ABSTRACT
The spliceosome is a complex small nuclear (sn)RNA-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. For each splicing event, the spliceosome is assembled de novo on a pre-mRNA substrate and a complex series of assembly steps leads to the active conformation. To comprehensively monitor pre-mRNA conformational dynamics during spliceosome assembly, we developed a strategy for single-molecule FRET (smFRET) that utilizes 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. In this chapter, we describe the identification of Ubc4 pre-mRNA that is efficiently spliced in vitro and the methods we have developed for the chemical synthesis of fluorescent Ubc4 pre-mRNA for smFRET.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Precursores de RNA
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Splicing de RNA
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Transferência Ressonante de Energia de Fluorescência
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article