Etude de cinétique de la traduction eucaryote à l'échelle de la molécule unique

Nicolas Fiszman 1
1 Laboratoire Charles Fabry / Biophotonique
LCF - Laboratoire Charles Fabry
Abstract : Protein synthesis is a central mechanism of cellular life and understanding it is a challenge in biomedical research. Single molecule studies permit each reactive system to be observed individually and provide access to asynchronous events difficult to observe in ensemble experiments, such as protein translation.This thesis presents the first results on single molecule eukaryotic (mammalian) translation. We observe the translational systems using fluorophores linked to oligonucleotides annealed to the RNA translated sequences. The observation of these fluorophores is done by total internal reflection fluorescence microscopy, the RNA being attached to a microscope slide. When reading the RNA, the ribosome unzips the fluorescent oligonucleotides and their departure times provide information about the position of the ribosome at different locations on the RNA strand. This method provides kinetic data on a large number of parallel translational systems that can be fitted using probability laws.With this method, we measure the in vitro kinetics of eukaryotic elongation and we reveal a delay due to a non-canonical initiation of the ribosome. Indeed, in our experiments, the ribosome is initially complexed on an RNA structure called Internal Ribosome Entry Site. In our experimental conditions, each incorporation of an amino acid in the nascent protein takes about one second while the IRES structure induces a delay of several tens of seconds on the first incorporation. These results open new perspectives for kinetic studies in more complex configurations such as the passage of the ribosome through RNA secondary structures.
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Nicolas Fiszman. Etude de cinétique de la traduction eucaryote à l'échelle de la molécule unique. Optique [physics.optics]. Institut d’Optique Graduate School, 2013. Français. ⟨NNT : 2013IOTA0001⟩. ⟨pastel-00939858⟩

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