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Transcription par une ARN Polymerase : mesures de forces à l'échelle de la molécule unique

Abstract : We have performed single molecule experiments to
measure the influence of mechanical force on a transcribing T7 RNA polymerase. This motor enzyme is highly processive, and structurally very related to Pol-I family including HIV 1 reverse transcriptase. One end of the template DNA is held by a polymerase anchored to a surface, the other end is attached to a microscopic bead which is captured by an optical trap that measures the force developed during transcription. We report velocity measurements performed with an applied force in the range 5-20 pN, and at different nucleotide concentrations. The nucleotide binding step in the catalytic cycle is limiting when lowering NTP concentration. We observe at low NTP concentration that the velocity of the enzyme becomes markedly force-dependent, whereas it is not at saturant NTP concentration. We deduce that the forward motion of the polymerase along DNA occurs upon nucleotide
binding in the beginning of the reactions cycle. The energy of NTP hydrolysis is thus not directly used as mechanical energy. Homologies among polymerases suggest that translocation mechanism is also shared. Thus we propose that for polymerases in general,
nucleotide binding is associated to forward motion.

We performed another type of single molecule
experiment by measuring the force during the mechanical opening of DNA. It allowed us to determine the rotationnal drag undergone by a rotating DNA molecule.

This DNA unzipping configuration has also been used to study the interaction between EcoR V protein and DNA. The dissociation energy shows a large variability linked to different affinities of the target sites of the enzyme.
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https://tel.archives-ouvertes.fr/tel-00011391
Contributor : Philippe Thomen <>
Submitted on : Monday, January 16, 2006 - 3:55:40 PM
Last modification on : Thursday, October 29, 2020 - 3:01:21 PM
Long-term archiving on: : Tuesday, April 6, 2010 - 4:39:06 PM

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  • HAL Id : tel-00011391, version 1

Citation

Philippe Thomen. Transcription par une ARN Polymerase : mesures de forces à l'échelle de la molécule unique. Biophysique [physics.bio-ph]. Université Pierre et Marie Curie - Paris VI, 2002. Français. ⟨tel-00011391⟩

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