Site-specific labeling of the ribosome for single-molecule spectroscopy. Academic Article uri icon

Overview

MeSH

  • Fluorescence Resonance Energy Transfer
  • Models, Molecular
  • Mutation
  • Protein Biosynthesis

MeSH Major

  • Fluorescent Dyes
  • Oligonucleotides
  • Ribosomes
  • Spectrometry, Fluorescence

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.

publication date

  • 2005

has subject area

  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Models, Molecular
  • Mutation
  • Oligonucleotides
  • Protein Biosynthesis
  • Ribosomes
  • Spectrometry, Fluorescence

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC546139

Digital Object Identifier (DOI)

  • 10.1093/nar/gki151

PubMed ID

  • 15647501

Additional Document Info

start page

  • 182

end page

  • 189

volume

  • 33

number

  • 1