title: A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis.
abstract: 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.
authors.affiliation: Department of Chemistry, Columbia University, New York, NY, USA.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Fei
authors.forename: Jiangning
authors.initials: J
authors.lastname: Wang
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Sternberg
authors.forename: Daniel D
authors.initials: DD
authors.lastname: MacDougall
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Elvekrog
authors.forename: Dileep K
authors.initials: DK
authors.lastname: Pulukkunat
authors.forename: Michael T
authors.initials: MT
authors.lastname: Englander
authors.forename: Ruben L
authors.initials: RL
authors.lastname: Gonzalez
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D012313:RNA