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Pince optique et microscopie de fluorescence pour l'étude de la synthèse des protéines en molécule unique

Antoine Le Gall 1 
1 Laboratoire Charles Fabry de l'Institut d'Optique / Biophotonique
LCFIO - Laboratoire Charles Fabry de l'Institut d'Optique
Abstract : We hereby report two approaches of the protein synthesis at the single molecule level. We use total internal reflection fluorescence microscopy to study the translation kinetic of two different types of ribosomes. The first ones, extracted from E. Coli (prokaryotic organism), are mutated in order to label them with a quantum dot (QD). The end of translation, which corresponds to the dissociation of the ribosome from the mRNA when the stop codon has been reached, is highlighted by the disparition of the QD from the surface. The second type of ribosome is extracted from rabbit cells (eukaryotic organism) and has not been modified (wild type), while a labeled oligonucleotide is hybridized on the mRNA. The helicase activity of the ribosome allowing the dissociation of two complementary strands, the oligonucleotide and so the label disappear at the same time while the ribosome moves along the mRNA and thus inform us about its activity. For these two types of ribosomes we measure their average translation speed in cell extracts.The second approach focuses on the properties of the mRNA, carrying the genetic code for the protein sequence. We developped an optical tweezer setup in order to manipulate and characterize the mechanical properties of nucleotides, as well as an original method to calibrate this optical trap. The consistency of our measurements with the litterature on the properties of a double stranded DNA will allow us to study secondary structures of mRNA.
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Submitted on : Saturday, December 3, 2011 - 3:12:33 PM
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  • HAL Id : tel-00647915, version 1



Antoine Le Gall. Pince optique et microscopie de fluorescence pour l'étude de la synthèse des protéines en molécule unique. Sciences agricoles. Université Paris Sud - Paris XI, 2011. Français. ⟨NNT : 2011PA112238⟩. ⟨tel-00647915⟩



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