Nucleic Acid Chaperone Activity Transforms the Energy Landscape of HIV-1 TAR RNA

Abstract

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle, including reverse transcription, where NC destabilizes the transactivation response RNA (TAR RNA) hairpin. To quantify the interaction of HIV-1 NC and TAR RNA, we use optical tweezers to exert tension upon the free ends of an individual TAR hairpin, forcing the hairpin open and then allowing it to close. We combine force-ramp experiments with a new analytical technique that quantitatively characterizes the energy landscape of equilibrium and non-equilibrium TAR hairpin unfolding and folding. We use the pulling rate dependence of the unfolding force to determine the distance to the transition state for this complex. This measurement reveals that to cause complete TAR unfolding, it is sufficient to unzip 10 bp of the 24 bp TAR hairpin stem in the absence of NC but only 5 bp of 24 in the presence of NC. Extrapolation of the measured TAR opening rate to zero pulling rate yields nearly 1000-fold faster opening with NC, equivalent to a decrease in the TAR opening barrier of 4.2 +/- 0.2 kcal/mol. Furthermore, equilibrium and complementary non-equilibrium TAR unfolding measurements using Crooks fluctuation theorem show that NC lowers the overall free energy of hairpin opening by 8.5 +/- 0.3 kcal/mol. These results demonstrate that NC destabilizes every nucleic acid base pair by strongly facilitating nucleic acid opening. This work was funded in part by Federal Funds from NCI, NIH under contract HHSN261200800001E (RJG).