Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA.
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.
Department of Physics, Northeastern University, Boston, MA 02115;
Micah J
MJ
McCauley
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455;
Ioulia
I
Rouzina
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455;
Kelly A
KA
Manthei
AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702;
Robert J
RJ
Gorelick
Department of Chemistry and Biochemistry, Center for Retroviral Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
Karin
K
Musier-Forsyth
Department of Physics, Northeastern University, Boston, MA 02115; mark@neu.edu.
Mark C
MC
Williams
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D059370:RNA Folding; D020871:RNA Stability; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048348:Reverse Transcription; D013816:Thermodynamics
D019590:Nucleocapsid Proteins; D012367:RNA, Viral
RNA binding; RNA stretching; force spectroscopy; single molecule