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

Sima Barhoom, Jaskiran Kaur, Barry S. Cooperman, Nechama I. Smorodinsky, Zeev Smilansky, Marcelo Ehrlich*, Orna Elroy-Stein

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
JournalNucleic Acids Research
Volume39
Issue number19
DOIs
StatePublished - Oct 2011

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