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

Cosa, Gonzalo; Harbron, Elizabeth J.; Zeng, Yining; Liu, Hsiao-Wei; O'Connor, Donald; Hosokawa, Chie; Musier‐Forsyth, Karin; Barbara, Paul F.

doi:10.1529/biophysj.104.043083

Cited by 66 publications

(164 citation statements)

References 37 publications

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“…4, as well as some additional stabilization of the singlestranded state by NC. In general, weakly base-paired G bases were recently shown to be sites for preferential NC binding accompanied by duplex destabilization in 2-aminopurine fluorescence studies (6), in single-molecule FRET studies (23), in SHAPE footprinting studies of HIV-1 NC (8) and MLV NC (24), and in a recent NMR study of MLV NC (25). Our results support this observation while also identifying the specific subset of these sites on TAR that are responsible for altering its unfolding landscape.…”

Section: ·Gmentioning

confidence: 99%

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

McCauley

Rouzina

Manthei

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.single molecule | force spectroscopy | RNA stretching | RNA binding T he transactivation response (TAR) RNA hairpin is a 59-nt sequence in the long-terminal repeat (LTR) of the HIV-1 genome that forms a 24-bp hairpin ( Fig. 1A) (1). This structure is essential in promoting viral transactivator protein (Tat)-mediated transcription. The protein-RNA complex further enhances LTR promoter activity (2). The highly stable TAR hairpin structure that stimulates viral RNA transcription becomes a liability during the early stage of a new infection, as TAR hairpins inhibit the minus-strand transfer step required for reverse transcription (1). To alleviate this inhibition, successful reverse transcription requires a key viral chaperone, the nucleocapsid (NC) protein. In vitro experiments have shown a 3,000-fold stimulation of the rate-limiting step of minus-strand transfer in the presence of NC (3), as NC is required to destabilize TAR RNA and the complementary repeat TAR DNA hairpin to allow subsequent strand annealing (1).HIV-1 NC is only 55 aa long, consisting of two highly conserved CCHC zinc fingers and a basic N terminus (1) (Fig. 1B). The multiple roles of NC during reverse transcription all use the same "chaperone" activity (1), which describes HIV-1 NC's ability to facilitate the rearrangement of nucleic acids into the most stable structures, with the lowest free energy (1). This chaperone activity is characterized by nucleic acid aggregation, duplex destabilization, and rapid kinetics of protein-nucleic acid interactions (3, 4). Aromatic residues in...

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Section: ·Gmentioning

confidence: 99%

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

McCauley

Rouzina

Manthei

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…[124][125][126][127][128][129][130][131][132][133][134][135][136] This chaperone activity, facilitating restructuring of the nucleic acid complex, has been attributed to both an aggregating ability of NC and a facility for duplex destabilization. 125,131,[137][138][139][140][141][142][143][144][145] This destabilization, however, is weak, 143,144,146,147 and will not completely melt DNA without a complementary strand. 6,138,148,149 Stretching and relaxation cycles can also reveal information on the kinetics of protein association and dissociation.…”

Section: Figure 11mentioning

confidence: 99%

Mechanisms of DNA binding determined in optical tweezers experiments

McCauley¹,

Williams²

2006

Biopolymers

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The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments. © 2006 Wiley Periodicals, Inc. Biopolymers 85:154–168, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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“…HTLV-1 is of interest because infection with this virus can lead to adult T-cell leukemia, as well as certain types of demyelinating diseases, such as tropical spastic paraparesis (1,6). The HIV-1 retrovirus has been widely studied, and it is known that both of these viruses contain many different structural and functional proteins (9,10,12,18,19,22,29), including the nucleocapsid proteins (NC), which are common in other retroviruses as well (11). In HIV-1, NC proteins play a critical role in chaperoning the strand transfer reactions during the reverse transcription process (27,36,45,48).…”

mentioning

confidence: 99%

“…Because one measures these dynamic changes one molecule at a time, it is possible to eliminate the averaging that occurs when all molecules are measured concurrently, as in traditional ensemble FRET. Time-resolved single-molecule spectroscopy has been used in previous studies to measure the nucleic acid conformational dynamics (9,10,35,37,38,54,55). Nucleic acid conformation changes in the presence of proteins have been observed using SMFRET (7).…”

mentioning

confidence: 99%

Human T-Cell Lymphotropic Virus Type 1 Nucleocapsid Protein-Induced Structural Changes in Transactivation Response DNA Hairpin Measured by Single-Molecule Fluorescence Resonance Energy Transfer

Darugar

Kim

Gorelick

et al. 2008

J Virol

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

Cited by 66 publications

References 37 publications

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Mechanisms of DNA binding determined in optical tweezers experiments

Human T-Cell Lymphotropic Virus Type 1 Nucleocapsid Protein-Induced Structural Changes in Transactivation Response DNA Hairpin Measured by Single-Molecule Fluorescence Resonance Energy Transfer

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