Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer

Yining Zeng, Hsiao Wei Liu, Christy F. Landes, Joo Kim Yoen, Xiaojing Ma, Yongjin Zhu, Karin Musier-Forsyth, Paul F. Barbara

Research output: Contribution to journalArticlepeer-review

Abstract

Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method. In this approach, an immobilized hairpin is exposed to a multistep programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer-only solutions. The sequence controllably "drags" single immobilized TAR hairpins among the kinetic stable states of the reaction mechanism; i.e., reactants, intermediates, and products. This single-molecule spectroscopy method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages along the reaction sequence, giving access to previously inaccessible kinetic processes and rate constants. By employing target oligonucleotides for specific TAR regions, we kinetically trap and investigate structural models for putative nucleation complexes for the annealing process. The new results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes. This includes information on the ability of NC to chaperone "reverse annealing" in single-strand transfer and the first observation of partially annealed, conformational substates in the annealing mechanism.

Original languageEnglish (US)
Pages (from-to)12651-12656
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number31
DOIs
StatePublished - Jul 31 2007
Externally publishedYes

Keywords

  • HIV-1 nucleocapsid protein
  • Transactivation response element

ASJC Scopus subject areas

  • General

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