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Nucleic Acids Research 2005 33(1):182-189; doi:10.1093/nar/gki151
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Published online 12 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 © Oxford University Press 2005; all rights reserved
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Article

Site-specific labeling of the ribosome for single-molecule spectroscopy

Magdalena Dorywalska1, Scott C. Blanchard1,2, Ruben L. Gonzalez, Jr1, Harold D. Kim2, Steven Chu2,* and Joseph D. Puglisi1,*

1 Department of Structural Biology, Stanford University School of Medicine Stanford, CA 94305-5126, USA 2 Department of Physics and Applied Physics, Stanford University Stanford, CA 94305-4060, USA

*To whom correspondence should be addressed. Tel: +1 650 498 4397; Fax: +1 650 723 8464; Email: puglisi{at}stanford.edu

Received October 11, 2004. Revised December 7, 2004. Accepted December 7, 2004.

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.

Correspondence may also be addressed to Steven Chu. Tel: +1 650 723 3571; Fax: +1 650 723 9173; Email: schu{at}LBL.gov


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