Conformational dynamics of single pre-mRNA molecules during in vitro splicing

被引：72

作者：

Abelson, John ^{[1
]}

Blanco, Mario ^{[2
]}

Ditzler, Mark A. ^{[3
,4
]}

Fuller, Franklin ^{[3
,4
]}

Aravamudhan, Pavithra ^{[3
,4
]}

Wood, Mona ^{[3
]}

Villa, Tommaso ^{[1
]}

Ryan, Daniel E. ^{[1
]}

Pleiss, Jeffrey A. ^{[1
]}

Maeder, Corina ^{[1
]}

Guthrie, Christine ^{[1
]}

Walter, Nils G. ^{[3
]}

机构：

[1] Univ Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USA

[2] Univ Michigan, Dept Cellular & Mol Biol, Ann Arbor, MI 48109 USA

[3] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA

[4] Univ Michigan, Dept Biophys, Single Mol Anal Grp, Ann Arbor, MI 48109 USA

来源：

NATURE STRUCTURAL & MOLECULAR BIOLOGY | 2010年 / 17卷 / 04期

基金：

美国国家卫生研究院;

关键词：

RIBOZYME MOLECULES; SPLICEOSOME; YEAST; CATALYSIS; IDENTIFICATION; PURIFICATION; MUTATIONS; SEQUENCES; FIDELITY; PATHWAY;

D O I：

10.1038/nsmb.1767

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：504 / U156

页数：11

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