Single Molecule Experiments on Nucleic Acid/Protein
Interactions in Chaperoned Nucleic Acid Rearrangements of HIV-1 Reverse
Transcription
Hsiao-Wei Liu1, Yining Zeng1,
Christy F. Landes1, Yoen Joo Kim1,
Yongjin Zhu1, Xiaojing Ma1, My-Nuong Vo2,
Karin Musier-Forsyth2, and Paul F. Barbara1
1Center for Nano and Molecular Science and Technology, The University of Texas at Austin, Austin, TX 78712, USA
2Department of Chemistry, The Ohio State University. 100 W. 18th Avenue, Columbus, Ohio 43210, University of Pennsylvania
Single molecule spectroscopy was used to investigate nucleocapsid protein (NC) "chaperoned" nucleic acid rearrangements in vitro that are relevant to the annealing of the DNA transactivation response element (TAR) hairpin to the complementary TAR RNA region of the viral genome, in the minus-strand transfer mechanism of HIV-1 reverse transcription. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level due to the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. By combining a flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy) we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing, demonstrating that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a "Y" shaped conformation and a closed one. We have also investigated the in vitro annealing mechanism by a novel multi-step SMS kinetic method, in which an immobilized hairpin is exposed to a multi-step programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer only solutions. This SMS method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages. The results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes, including the first information on the ability of NC to chaperone "reverse annealing" in minus strand transfer and the first observation of partially annealed, conformational sub-states in the annealing mechanism.