Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA
Genre
Journal ArticleDate
2011-10-01Author
Barhoom, SKaur, J
Cooperman, BS
Smorodinsky, NI
Smilansky, Z
Ehrlich, M
Elroy-Stein, O
Subject
AnimalsAstrocytes
CHO Cells
Cricetinae
Cricetulus
Fluorescence Resonance Energy Transfer
Fluorescent Dyes
Mice
Mice, Inbred C57BL
Microscopy, Fluorescence
Protein Biosynthesis
RNA, Transfer
Single-Cell Analysis
Viral Proteins
Permanent link to this record
http://hdl.handle.net/20.500.12613/5509
Metadata
Show full item recordDOI
10.1093/nar/gkr601Abstract
We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection. © The Author(s) 2011. Published by Oxford University Press.Citation to related work
Oxford University Press (OUP)Has part
Nucleic Acids ResearchADA compliance
For Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact scholarshare@temple.eduae974a485f413a2113503eed53cd6c53
http://dx.doi.org/10.34944/dspace/5491