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Mechanical unfolding of two DIS RNA kissing complexes from HIV-1.

ABSTRACT: An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg 2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 +/- 4 to 46 +/- 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of deltaX(++), which is the distance from the folded structure to the unfolding transition state. The deltaX(++) of Mal is approximately 0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg 2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg 2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.

SUBMITTER: Li PT

PROVIDER: S-EPMC2768397 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Mechanical unfolding of two DIS RNA kissing complexes from HIV-1.

Li Pan T X PT Tinoco Ignacio I

Journal of molecular biology 20090301 5

An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg 2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either ...[more]

PMID: 19452632

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Project description:Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force (f), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. For simple hairpins, such as P5GA, application of constant force or constant loading rate results in bistable cooperative transitions between folded and unfolded states without populating any intermediates. The transition state location (DeltaxFTS) changes dramatically as the loading rate is varied. At loading rates comparable to those used in laser optical tweezer experiments, the hairpin is plastic, with DeltaxFTS being midway between folded and unfolded states; whereas at high loading rates, DeltaxFTS moves close to the folded state, i.e., RNA is brittle. For the 29-nucleotide TAR RNA with the three-nucleotide bulge, unfolding occurs in a nearly two-state manner with an occasional pause in a high free energy metastable state. Forced unfolding of the 55 nucleotides of the Hepatitis IRES domain IIa, which has a distorted L-shaped structure, results in well-populated stable intermediates. The most stable force-stabilized intermediate represents straightening of the L-shaped structure. For these structures, the unfolding pathways can be predicted using the contact map of the native structures. Unfolding of a RNA motif with internal multiloop, namely, the 109-nucleotide prohead RNA that is part of the 29 DNA packaging motor, at constant value of rf occurs with three distinct rips that represent unraveling of the paired helices. The rips represent kinetic barriers to unfolding. Our work shows 1), the response of RNA to force is largely determined by the native structure; and 2), only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.

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Mechanical unfolding of two DIS RNA kissing complexes from HIV-1.

Publications

Mechanical unfolding of two DIS RNA kissing complexes from HIV-1.

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