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Methods. 2018 Mar 15;137:37-48. doi: 10.1016/j.ymeth.2017.12.007. Epub 2017 Dec 13.

Ensemble and single-molecule FRET studies of protein synthesis.

Author information

1
Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, United States.
2
Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, United States. Electronic address: Dmitri_Ermolenko@urmc.rochester.edu.

Abstract

Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges.

KEYWORDS:

Ensemble FRET; Fluorescence; Labeling; Ribosome; Single-molecule FRET

PMID:
29247758
PMCID:
PMC5866757
DOI:
10.1016/j.ymeth.2017.12.007
[Indexed for MEDLINE]
Free PMC Article

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