TY - JOUR
T1 - Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA
AU - Barhoom, Sima
AU - Kaur, Jaskiran
AU - Cooperman, Barry S.
AU - Smorodinsky, Nechama I.
AU - Smilansky, Zeev
AU - Ehrlich, Marcelo
AU - Elroy-Stein, Orna
N1 - Funding Information:
Funding for open access charge: Anima Cell Metrology research grant (to O.E.S. and M.E.), National Institutes of Health (grants No. GM090404-01 and GM-071014 to B.C.), BARD (grant No. 15-4192-09 to M.E.) and the Legacy Heritage Biomedical Science Partnership Program of the Israel Science Foundation (grant No. 1911/08 to O.E.S.).
PY - 2011/10
Y1 - 2011/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=80455168326&partnerID=8YFLogxK
U2 - 10.1093/nar/gkr601
DO - 10.1093/nar/gkr601
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AN - SCOPUS:80455168326
SN - 0305-1048
VL - 39
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 19
ER -