Specific Binding of the Nucleocapsid Protein Transforms the Folding Landscape of the HIV-1 TAR RNA Hairpin

Abstract

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC destabilizes nucleic acids, including the transactivation response (TAR) hairpin to facilitate their structural re-arrangement into the lowest free energy conformations. By combining single molecule optical tweezers measurements with mfold-based free energy calculations of the unfolding landscape, we determine the equilibrium TAR stability and characterize the unfolding transition state in both the presence and absence of NC. Our results show that protein binding occurs specifically at guanines that border structural defects in the upper part of TAR hairpin. This results in preferential destabilization by NC of this part of TAR, thereby shifting the transition state closer to the bottom of the TAR stem, leading to a shorter critical unfolding length. Thus, NC facilitates TAR RNA annealing to its complementary DNA hairpin while having little effect on the stability of the final annealed duplexes. These results provide the first direct measurement of alterations in an RNA folding landscape that are induced by protein-RNA interactions, and are a novel case study of the effect of a protein that specifically destabilizes particular elements of the nucleic acid secondary structure.