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    Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

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    Name:
    Quantitative single cell monitoring ...
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    Genre
    Journal Article
    Date
    2011-10-01
    Author
    Barhoom, S
    Kaur, J
    Cooperman, BS
    Smorodinsky, NI
    Smilansky, Z
    Ehrlich, M
    Elroy-Stein, O
    Subject
    Animals
    Astrocytes
    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
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    Permanent link to this record
    http://hdl.handle.net/20.500.12613/5509
    
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    DOI
    10.1093/nar/gkr601
    Abstract
    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 Research
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    ae974a485f413a2113503eed53cd6c53
    http://dx.doi.org/10.34944/dspace/5491
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