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Biophys. J. BioFAST: First Published August 12, 2005. doi:10.1529/biophysj.105.065326
© 2005 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2005.
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Hsiao-Wei Liu
Gonzalo Cosa
Christy F. Landes
Yining Zeng
Brandie J. Kovaleski
Daniel G. Mullen
George Barany
Karin Musier-Forsyth
Paul F. Barbara
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid Protein Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

Hsiao-Wei Liu 1, Gonzalo Cosa 1, Christy F. Landes 1, Yining Zeng 1, Brandie J. Kovaleski 2, Daniel G. Mullen 2, George Barany 2, Karin Musier-Forsyth 2 and Paul F. Barbara 1*

1 Department of Chemistry and Biochemistry, University of Texas, Austin
2 Department of Chemistry, University of Minnesota

* To whom correspondence should be addressed. E-mail: p.barbara{at}mail.utexas.edu.

Submitted on April 25, 2005
Revised on June 9, 2005
Accepted on 3 August 2005


  Abstract
The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3'/5' termini, termed the "zipper" complex, or nucleation through the hairpin loops in a "kissing" complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.

Key Words: Fluorescence resonance energy transfer, Single-molecule spectroscopy, TAR DNA:RNA annealing




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Copyright © 2005 by the Biophysical Society.