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- W2259260215 abstract "We developed a system using fluorescent proteins to analyze gene expression in real-time, and to follow individual mRNPs. An array of genes coding for a functional mRNA that contains 24 repeats of the MS2 coat protein binding motif combined with the MS2 coat protein fused to GFP or YFP allowed us to analyze the kinetics of transcription in real time and to detect single molecules of RNA in live cells. In these studies we used photo-bleaching of GFP-labeled mRNAs and of a YFP-pol II fusion protein and photoactivation of paGFP labeled mRNA. Analysis of the complex process of transcription using fluorescent polymerase as well as fluorescent MS2 proteins provided an opportunity to model the kinetic steps of RNA synthesis. These results yield rate constants for each of the steps of promoter assembly, initiation and elongation. They demonstrate that transcription is inefficient and that polymerases can elongate faster than thought, but can pause stochastically. RNAs can then be followed as single molecules in the nucleus and cytoplasm. The analysis demonstrated that subsequent RNA movements were not directed, but governed by rules of simple diffusion. The kinetics of their transport through nuclear pores yielded a biexponential kinetics. Once in the cytoplasm, mRNAs diffuse but also can be directed to their destinations by virtue of a zipcode motif in the RNA. A zipcode binding protein (ZBP1) is essential for this localization and inhibition of translation. β-actin can thusly be synthesized at sites of active F-actin polymerization in migrating fibroblasts or growing neurons. Supported by NIH-EB, GM." @default.
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- W2259260215 date "2009-04-01" @default.
- W2259260215 modified "2023-10-16" @default.
- W2259260215 title "Imaging Real‐Time Gene Expression in Living Cells" @default.
- W2259260215 doi "https://doi.org/10.1096/fasebj.23.1_supplement.316.3" @default.
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