Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Micah J. McCauley, Ioulia Rouzina, Kelly A. Manthei, Robert J. Gorelick, Karin Musier-Forsyth, Mark C. Williams

Research output: Contribution to journalArticlepeer-review

24 Scopus citations


Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.

Original languageEnglish (US)
Pages (from-to)13555-13560
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number44
StatePublished - Nov 3 2015


  • Force spectroscopy
  • RNA binding
  • RNA stretching
  • Single molecule


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