Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.
Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.
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DOI：
10.1093/nar/gkr601
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发表时间：
2011-10
影响因子：
14.9
通讯作者：
Elroy-Stein O
中科院分区：
文献类型：
--
作者：
Barhoom S;Kaur J;Cooperman BS;Smorodinsky NI;Smilansky Z;Ehrlich M;Elroy-Stein O
我们开发了一种利用荧光tRNA进行翻译监测的新技术(FtTM)。FtTM通过在D-loop(fl-tRNA)中荧光标记的转基因未带电tRNA(fl-tRNA)的定量显微镜，能够在亚微米分辨率下识别和监测活细胞内的活性蛋白质合成位点。通过与细胞因子的共定位和蛋白质合成抑制时的动态变化，证实了fl-tRNA定位于活跃的翻译位点。此外，当分别标记为FRET对的fl-tRNAs占据核糖体上的相邻位置时产生的荧光共振能量转移(FRET)信号，定量地反映了特定细胞区域的蛋白质合成水平。此外，FRET信号能够检测到蛋白质合成活动的种群内可变性。我们证明，FtTM可以定量比较不同细胞类型之间的蛋白质合成，监测抗生素和应激剂的影响，并表征病毒感染时蛋白质合成空间区隔的变化。
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.
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影响因子：
6.7
作者：
Castelló A;Quintas A;Sánchez EG;Sabina P;Nogal M;Carrasco L;Revilla Y
通讯作者：
Revilla Y
影响因子：
64.8
作者：
通讯作者：
--
DOI：
10.1046/j.1432-1033.2002.02974.x
发表时间：
2002-06-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Gerlitz, G;Jagus, R;Elroy-Stein, O
通讯作者：
Elroy-Stein, O
影响因子：
4.5
作者：
Betteridge, Thu;Liu, Hanqing;Hou, Ya-Ming
通讯作者：
Hou, Ya-Ming
影响因子：
5
作者：
Anthony, S. J.;Maan, N.;Mertens, P. P. C.
通讯作者：
Mertens, P. P. C.