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Published September 28, 2011 | Accepted Version + Supplemental Material
Journal Article Open

Site-Specific Fluorescent Labeling of Nascent Proteins on the Translating Ribosome


As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosomenascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

Additional Information

© 2011 American Chemical Society. Received: July 15, 2011. Publication Date (Web): August 26, 2011. We thank Prof. Swartz (Stanford University) for help with the generation of S30 extracts; Prof. Schultz (TSRI) for providing the pEVOL plasmid containing the tRNACUA/aaRS pair; Prof. Chapman (TSRI) for a sample of Cm; Profs. Tirrell, Barton, and Dougherty (Caltech) for use of their facilities; Prof. Sando(Kyushu University) for the RF-1 inhibitor aptamer sequence; and D. Dougherty and members of the Shan group for helpful comments on the manuscript. This work was supported by NIH Grant GM078024 to S.S. S.S. was supported by the Henry Dreyfus Teacher-Scholar Award, the Beckman Young Investigator Award, and the Packard and Lucile Award in Science and Engineering.

Attached Files

Accepted Version - nihms323508.pdf

Supplemental Material - ja206626g_si_001.pdf


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