Paul F Barbara — Page 2

PubMed2004

Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein.

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.

Binding Sites↗

PubMed2003

Nucleic acid conformational changes essential for HIV-1 nucleocapsid protein-mediated inhibition of self-priming in minus-strand transfer.

Reverse transcription of the HIV-1 genome is a complex multi-step process. HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein that has been shown to greatly facilitate the nucleic acid rearrangements that precede the minus-strand transfer step in reverse transcription. NC destabilizes the highly structured transactivation response region (TAR) present in the R region of the RNA genome, as well as a complementary hairpin structure ("TAR DNA") at the 3'-end of the newly synthesized minus-strand strong-stop DNA ((-) SSDNA). Melting of the latter structure inhibits a self-priming (SP) reaction that competes with the strand transfer reaction. In an in vitro minus-strand transfer system consisting of a (-) SSDNA mimic and a TAR-containing acceptor RNA molecule, we find that when both nucleic acids are present, NC facilitates formation of the transfer product and the SP reaction is greatly reduced. In contrast, in the absence of the acceptor RNA, NC has only a small inhibitory effect on the SP reaction. To further investigate NC-mediated inhibition of SP, we developed a FRET-based assay that allows us to directly monitor conformational changes in the TAR DNA structure upon NC binding. Although the majority ( approximately 71%) of the TAR DNA molecules assume a folded hairpin conformation in the absence of NC, two minor "semi-folded" and "unfolded" populations are also observed. Upon NC binding to the TAR DNA alone, we observe a modest shift in the population towards the less-folded states. In the presence of the RNA acceptor molecule, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These measurements help to explain why acceptor RNA is required for significant inhibition of the SP reaction by NC, and support the hypothesis that NC-mediated annealing of nucleic acids is a concerted process wherein the unwinding step occurs in synchrony with hybridization.

DNA, Viral↗