title: Site-specific labeling of the ribosome for single-molecule spectroscopy.
abstract: Single-molecule fluorescence spectroscopy can reveal mechanistic and kinetic details that may not be observed in static structural and bulk biochemical studies of protein synthesis. One approach requires site-specific and stable attachment of fluorophores to the components of translation machinery. Fluorescent tagging of the ribosome is a prerequisite for the observation of dynamic changes in ribosomal conformation during translation using fluorescence methods. Modifications of the ribosomal particle are difficult due to its complexity and high degree of sequence and structural conservation. We have developed a general method to label specifically the prokaryotic ribosome by hybridization of fluorescent oligonucleotides to mutated ribosomal RNA. Functional, modified ribosomes can be purified as a homogenous population, and fluorescence can be monitored from labeled ribosomal complexes immobilized on a derivatized quartz surface.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Dorywalska
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
authors.forename: Ruben L
authors.initials: RL
authors.lastname: Gonzalez
authors.forename: Harold D
authors.initials: HD
authors.lastname: Kim
authors.forename: Steven
authors.initials: S
authors.lastname: Chu
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry*; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides