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Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

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  • Nancy Beerens
  • ,
  • Mette D E Jepsen, Department of Molecular Biology, Interdisciplinary Nanoscience Center, Aarhus University, Danmark
  • Volodymyr Nechyporuk-Zloy, Interdisciplinary Nanoscience Center, Aarhus University, Danmark
  • Asger C Krüger, Danmark
  • Jean-Luc Darlix, UMR 7213 CNRS, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, Frankrig
  • Jørgen Kjems
  • Victoria Birkedal
HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription.
OriginalsprogEngelsk
TidsskriftRNA
Vol/bind19
Nummer4
Sider (fra-til)517-26
Antal sider10
ISSN1355-8382
DOI
StatusUdgivet - apr. 2013

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