title: Preparation of ribosomes for smFRET studies: A simplified approach.
abstract: During the past decade, single-molecule studies of the ribosome have significantly advanced our understanding of protein synthesis. The broadest application of these methods has been towards the investigation of ribosome conformational dynamics using single-molecule Förster resonance energy transfer (smFRET). The recent advances in fluorescently labeled ribosomes and translation components have resulted in success of smFRET experiments. Various methods have been employed to target fluorescent dyes to specific locations within the ribosome. Primarily, these methods have involved additional steps including subunit dissociation and/or full reconstitution, which could result in ribosomes of reduced activity and translation efficiency. In addition, substantial time and effort are required to produce limited quantities of material. To enable rapid and large-scale production of highly active, fluorescently labeled ribosomes, we have developed a procedure that combines partial reconstitution with His-tag purification. This allows for a homogeneous single-step purification of mutant ribosomes and subsequent integration of labeled proteins. Ribosomes produced with this method are shown to be as active as ribosomes purified using classical methods. While we have focused on two labeling sites in this report, the method is generalizable and can in principle be extended to any non-essential ribosomal protein.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA.
authors.forename: Bassem
authors.initials: B
authors.lastname: Shebl
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA.
authors.forename: Drew E
authors.initials: DE
authors.lastname: Menke
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Pennella
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA.
authors.forename: Raghav R
authors.initials: RR
authors.lastname: Poudyal
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA.
authors.forename: Donald H
authors.initials: DH
authors.lastname: Burke
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA. Electronic address: cornishp@missouri.edu.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Cornish
mesh_terms: D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D020653:Peptide Elongation Factor G / Q000737:chemistry*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D006639:Histidine; D013395:Sucrose; D004247:DNA; D012343:RNA, Transfer
keywords: Fluorescence; Purification; Ribosome; smFRET