A HIGHLY PURIFIED, FLUORESCENTLY LABELED IN VITRO TRANSLATION SYSTEM FOR SINGLE-MOLECULE STUDIES OF PROTEIN SYNTHESIS

被引：33

作者：

Fei, Jingyi ^{[1
]}

Wang, Jiangning ^{[1
]}

Sternberg, Samuel H. ^{[1
]}

MacDougall, Daniel D. ^{[1
]}

Elvekrog, Margaret M. ^{[1
]}

Pulukkunat, Dileep K. ^{[1
]}

Englander, Michael T. ^{[1
,2
]}

Gonzalez, Ruben L., Jr. ^{[1
]}

机构：

[1] Columbia Univ, Dept Chem, New York, NY 10027 USA

[2] Columbia Univ, Integrated Program Cellular Mol & Biomed Sci, New York, NY 10027 USA

来源：

METHODS IN ENZYMOLOGY, VOL 472: SINGLE MOLECULE TOOLS, PT A: FLUORESCENCE BASED APPROACHES | 2010年 / 472卷

关键词：

PEPTIDE-BOND FORMATION; AMINOACYL-TRANSFER-RNA; ELONGATION-FACTOR TU; RELEASE-FACTORS RF1; RIBOSOMAL L1 STALK; GTPASE ACTIVATION; INITIATION; DYNAMICS; TERMINATION; SELECTION;

D O I：

10.1016/S0076-6879(10)72008-5

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful tool for mechanistic investigations of increasingly complex biochemical systems. Recently, we and others have successfully used smFRET to directly investigate the role of structural dynamics in the function and regulation of the cellular protein synthesis machinery. A significant challenge to these experiments, and to analogous experiments in similarly complex cellular machineries, is the need for specific and efficient fluorescent labeling of the biochemical system at locations that are both mechanistically informative and minimally perturbative to the biological activity. Here, we describe the development of a highly purified, fluorescently labeled in vitro translation system that we have successfully designed for smFRET studies of protein synthesis. The general approaches we outline should be amenable to single-molecule fluorescence studies of other complex biochemical systems.

引用

页码：221 / 259

页数：39