Purification and electron microscopic visualization of functional human spliceosomes

被引：55

作者：

Zhou, ZL

Sim, JG

Griffith, J

Reed, R ^{[1
]}

机构：

[1] Harvard Univ, Sch Med, Dept Cell Biol, Boston, MA 02115 USA

[2] Harvard Univ, Dept Mol & Cellular Biol, Cambridge, MA 02138 USA

[3] Univ N Carolina, Lineberger Comprehens Canc Ctr, Chapel Hill, NC 27599 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2002年 / 99卷 / 19期

关键词：

D O I：

10.1073/pnas.182427099

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Pre-mRNA splicing takes place in a large and highly dynamic complex known as the spliceosome. Here we report the optimization of a maltose-binding protein (MBP) affinity-purification method to isolate functional spliceosomes for electron microscopic analysis. Visualization of the spliceosome preparations revealed distinct 40-60 nm particles. Immunogold-conjugated antibodies to spliceosome components specifically label these particles, which are eliminated by treatment with either RNase or protease. Moreover, spliceosomes assembled on two different pre-mRNAs are indistinguishable. This first visualization of purified functional spliceosomes assembled in vitro reveals striking structural features, including one or more central cavities and multiple elongate lobes.

引用

页码：12203 / 12207

页数：5

共 24 条

[1] The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several US-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein [J].