Secondary Structure and Secondary Structure Dynamics of DNA Hairpins Complexed with HIV-1 NC Protein

Abstract

Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. We report herein single-molecule fluorescence resonance energy transfer measurements on single immobilized TAR DNA hairpins and hairpin mutants complexed with NC (i.e., TAR DNA/NC). Using this approach we have explored the conformational distribution and dynamics of the hairpins in the presence and absence of NC protein. The data demonstrate that NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully “closed” conformation to essentially one specific “partially open” conformation. In this specific conformation, the two terminal stems are “open” or unwound and the other stems are closed. This partially open conformation is arguably a key TAR DNA intermediate in the NC-induced annealing mechanism of TAR DNA.