Mechanical unfolding of RNA hairpins

Mechanical unfolding trajectories, generated by applying constant force in optical tweezer experiments, show that RNA hairpins and the P5abc subdomain of the group I intron unfold reversibly. We use coarse-grained Go-like models for RNA hairpins to explore forced-unfolding over a broad range of temperatures. A number of predictions that are amenable to experimental tests are made. At the critical force the hairpin jumps between folded and unfolded conformations without populating any discernible intermediates. The phase diagram in the force-temperature (f,T) plane shows that the hairpin unfolds by an all-or-none process. The cooperativity of the unfolding transition increases dramatically at low temperatures. Free energy of stability, obtained from time averages of mechanical unfolding trajectories, coincide with ensemble averages which establishes ergodicity. The hopping time between the the native basin of attraction (NBA) and the unfolded basin increases dramatically along the phase boundary. Thermal unfolding is stochastic whereas mechanical unfolding occurs in "quantized steps" with great variations in the step lengths. Refolding times, upon force quench, from stretched states to the NBA is "at least an order of magnitude" greater than folding times by temperature quench. Upon force quench from stretched states the NBA is reached in at least three stages. In the initial stages the mean end-to-end distance decreases nearly continuously and only in the last stage there is a sudden transition to the NBA. Because of the generality of the results we propose that similar behavior should be observed in force quench refolding of proteins.Comment: 23 pages, 6 Figures. in press (Proc. Natl. Acad. Sci.

Electrostatic free energy landscapes for nucleic acid helix assembly

Metal ions are crucial for nucleic acid folding. From the free energy landscapes, we investigate the detailed mechanism for ion-induced collapse for a paradigm system: loop-tethered short DNA helices. We find that Na(+) and Mg(2+) play distinctive roles in helix–helix assembly. High [Na(+)] (>0.3 M) causes a reduced helix–helix electrostatic repulsion and a subsequent disordered packing of helices. In contrast, Mg(2+) of concentration >1 mM is predicted to induce helix–helix attraction and results in a more compact and ordered helix–helix packing. Mg(2+) is much more efficient in causing nucleic acid compaction. In addition, the free energy landscape shows that the tethering loops between the helices also play a significant role. A flexible loop, such as a neutral loop or a polynucleotide loop in high salt concentration, enhances the close approach of the helices in order to gain the loop entropy. On the other hand, a rigid loop, such as a polynucleotide loop in low salt concentration, tends to de-compact the helices. Therefore, a polynucleotide loop significantly enhances the sharpness of the ion-induced compaction transition. Moreover, we find that a larger number of helices in the system or a smaller radius of the divalent ions can cause a more abrupt compaction transition and a more compact state at high ion concentration, and the ion size effect becomes more pronounced as the number of helices is increased

Capturing the essence of folding and functions of biomolecules using Coarse-Grained Models

The distances over which biological molecules and their complexes can function range from a few nanometres, in the case of folded structures, to millimetres, for example during chromosome organization. Describing phenomena that cover such diverse length, and also time scales, requires models that capture the underlying physics for the particular length scale of interest. Theoretical ideas, in particular, concepts from polymer physics, have guided the development of coarse-grained models to study folding of DNA, RNA, and proteins. More recently, such models and their variants have been applied to the functions of biological nanomachines. Simulations using coarse-grained models are now poised to address a wide range of problems in biology.Comment: 37 pages, 8 figure

Dbpa Is A Region-Specific Rna Helicase

DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core\u27s grasp. In other words, it is not known if DbpA is a site-specific enzyme or region-specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region-specific or a site-specific enzyme. Our data suggest that DbpA is a region-specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C-terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double-helices in its vicinity. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core\u27s grasp