A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand

  • Sun Q
  • Phan J
  • Friberg A
 et al. 
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Abstract

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

Author-supplied keywords

  • Cysteine tethering
  • Fragment-based drug discovery
  • K-Ras
  • Second-site screen

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Authors

  • Qi Sun

  • Jason Phan

  • Anders R. Friberg

  • Demarco V. Camper

  • Edward T. Olejniczak

  • Stephen W. Fesik

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