Journal
NUCLEIC ACIDS RESEARCH
Volume 33, Issue 1, Pages 182-189Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gki151
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Funding
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM051266] Funding Source: NIH RePORTER
- NIGMS NIH HHS [GM51266, R01 GM051266] Funding Source: Medline
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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.
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