Unusual mechanical stability of a minimal RNA kissing complex.
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ABSTRACT:
SUBMITTER: Li PT
PROVIDER: S-EPMC1635091 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An RNA kissing loop from the Moloney murine leukemia virus (MMLV) exhibits unusual mechanical stability despite having only two intermolecular base pairs. Mutations at this junction have been shown to destabilize genome dimerization, with concomitant reductions in viral packaging efficiency and infectivity. Optical tweezers experiments have shown that it requires as much force to break the MMLV kissing-loop complex as is required to unfold an 11-bp RNA hairpin [Li PTX, Bustamante C, Tinoco I (2006) Proc Natl Acad Sci USA 103:15847-15852]. Using nonequilibrium all-atom molecular dynamics simulations, we have developed a detailed model for the kinetic intermediates of the force-induced dissociation of the MMLV dimerization initiation site kissing loop. Two hundred and eight dissociation events were simulated (approximately 16 ?s total simulation time) under conditions of constant applied external force, which we use to construct a Markov state model for kissing-loop dissociation. We find that the complex undergoes a conformational rearrangement, which allows for equal distribution of the applied force among all of the intermolecular hydrogen bonds, which is intrinsically more stable than the sequential unzipping of an ordinary hairpin. Stacking interactions with adjacent, unpaired loop adenines further stabilize the complex by increasing the repair rate of partially broken H-bonds. These stacking interactions are prominently featured in the transition state, which requires additional coordinates orthogonal to the end-to-end extension to be uniquely identified. We propose that these stabilizing features explain the unusual stability of other retroviral kissing-loop complexes such as the HIV dimerization site.
| S-EPMC3386141 | biostudies-literature
Project description:We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ~3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.
| S-EPMC3222126 | biostudies-literature
Project description: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.
| S-EPMC2768397 | biostudies-literature
Project description:The dimerization of viral RNA through noncovalent interactions at their 5' ends is a key step in the life cycle of retroviruses. In Moloney murine leukemia virus, three stem-loops are important in this process. One is a self-complementary tetraloop (H1), but the other two stem-loops (H2, H3) contain highly conserved GACG tetraloops that are not self-complementary sequences. Using two-dimensional NMR, we determined the structure of the H3 stem-loop. Surprisingly, it forms a stable, homodimeric kissing complex through only two intermolecular G small middle dotC base pairs. Cross-strand interactions of the adenines adjacent to the intermolecular G small middle dotC base pairs, plus unusual strong electrostatic interactions around the base pairs, contribute to the unexpected stability. This structure shows how even stem-loops without self-complementary sequences can facilitate the intermolecular recognition between two identical RNAs, and thus initiate dimerization and encapsidation of retroviral RNAs.
| S-EPMC16875 | biostudies-literature
Project description:Two identical 5'GACG3' tetra-loop motifs with different stem sequences (called H2 and H3) are found in the 5' end region of Moloney Murine Leukemia Virus (MMLV) genomic RNA. They play important roles in RNA dimerization and encapsidation through two identical tetra-loops (5'GACG3') forming a loop-to-loop kissing complex, the smallest RNA kissing complex ever found in nature. We examined the effects of a loop-closing base pair as well as a stem sequence on the conformational stability of the kissing complex. UV melting analysis and gel electrophoresis were performed on eight RNA sequences mimicking the H2 and H3 hairpin tetra-loops with variation in loop-closing base pairs. Our results show that changing the loop-closing base pair from the wildtype (5'A·U3' for H3, 5'U·A3' for H2) to 5'G·C3'/5'C·G3' has significant effect on the stability of the kissing complexes: the substitution to 5'C·G3' significantly decreases both thermal and mechanical stability, while switching to the 5'G·C3' significantly increases the mechanical stability only. The kissing complexes with the wildtype loop-closing base pairs (5'A·U3' for H3 and 5'U·A3' for H2) show different stability when attached to a different stem sequence (H2 stem vs. H3 stem). This suggests that not only the loop-closing base pair itself, but also the stem sequence, affects the conformational stability of the RNA kissing complex.
| S-EPMC5268073 | biostudies-literature
Project description:RNA kissing complexes are essential for genomic RNA dimerization and regulation of gene expression, and their structures and stability are critical to their biological functions. In this work, we used our previously developed coarse-grained model with an implicit structure-based electrostatic potential to predict three-dimensional (3D) structures and stability of RNA kissing complexes in salt solutions. For extensive RNA kissing complexes, our model shows great reliability in predicting 3D structures from their sequences, and our additional predictions indicate that the model can capture the dependence of 3D structures of RNA kissing complexes on monovalent/divalent ion concentrations. Moreover, the comparisons with extensive experimental data show that the model can make reliable predictions on the stability for various RNA kissing complexes over wide ranges of monovalent/divalent ion concentrations. Notably, for RNA kissing complexes, our further analyses show the important contribution of coaxial stacking to the 3D structures and stronger stability than the corresponding kissing-interface duplexes at high salts. Furthermore, our comprehensive analyses for RNA kissing complexes reveal that the thermally folding pathway for a complex sequence is mainly determined by the relative stability of two possible folded states of kissing complex and extended duplex, which can be significantly modulated by its sequence.
| S-EPMC6795135 | biostudies-literature
Project description:In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5'-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.
| S-EPMC3664431 | biostudies-literature
Project description:The ?-catenin/?-catenin complex serves as a critical molecular interface involved in cadherin-catenin-based mechanosensing at the cell-cell adherence junction that plays a critical role in tissue integrity, repair, and embryonic development. This complex is subject to tensile forces due to internal actomyosin contractility and external mechanical micro-environmental perturbation. However, the mechanical stability of this complex has yet to be quantified. Here, we directly quantified the mechanical stability of the ?-catenin/?-catenin complex and showed that it has enough mechanical stability to survive for tens to hundreds of seconds within physiological level of forces up to 10?pN. Phosphorylation or phosphotyrosine-mimetic mutations (Y142E or/and T120E) on ?-catenin shorten the mechanical lifetime of the complex by tens of fold over the same force range. These results provide insights into the regulation of the ?-catenin/?-catenin complex by phosphorylation.
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Project description:RNA-RNA interactions are essential for genomic RNA dimerization, mRNA splicing, and many RNA-related gene expression and regulation processes. The prediction of the structure and folding stability of RNA-RNA complexes is a problem of significant biological importance and receives substantial interest in the biological community. The VfoldCPX server provides a new web interface to predict the two-dimensional (2D) structures of RNA-RNA complexes from the nucleotide sequences. The VfoldCPX server has several novel advantages including the ability to treat RNAs with tertiary contacts (crossing base pairs) such as loop-loop kissing interactions and the use of physical loop entropy parameters. Based on a partition function-based algorithm, the server enables prediction for structure with and without tertiary contacts. Furthermore, the server outputs a set of energetically stable structures, ranked by their stabilities. The results allow users to gain extensive physical insights into RNA-RNA interactions and their roles in RNA function. The web server is freely accessible at "http://rna.physics.missouri.edu/vfoldCPX".
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Project description:Mechanical metamaterials that are engineered with sub-unit structures present unusual mechanical properties depending on the loading direction. Although they show promise, their practical utility has so far been somewhat limited because, to the best of our knowledge, no study about the potential of mechanical metamaterials made from sophisticatedly tailored sub-unit structures has been made. Here, we present a mechanical metamaterial whose mechanical properties can be systematically designed without changing its chemical composition or weight. We study the mechanical properties of triply periodic bicontinuous structures whose detailed sub-unit structure can be precisely fabricated using various sub-micron fabrication methods. Simulation results show that the effective wave velocity of the structures along with different directions can be designed to introduce the anisotropy of stiffness by changing a volume fraction and aspect ratio. The ratio of Young's modulus to shear modulus can be increased by up to at least 100, which is a 3500% increase over that of isotropic material (2.8, acrylonitrile butadiene styrene). Furthermore, Poisson's ratio of the constituent material changes the ratio while Young's modulus does not influence it. This study presents the promising potential of mechanical metamaterials for versatile industrial and biomedical applications.
| S-EPMC4738250 | biostudies-literature