Loop and stem dynamics during RNA hairpin folding and unfolding

Sarkar, K. R.; Nguyễn, Duy Anh; Gruebele, Martin

doi:10.1261/rna.2253310

Cited by 36 publications

(62 citation statements)

References 26 publications

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“…Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). However, due to the complexity of the RNA folding energy landscape (37)(38)(39)(40)(41)(42)(43)(44)(45)(46) and the limitations of experimental tools (47)(48)(49)(50)(51)(52)(53)(54)(55), many fundamental problems, ...…”

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confidence: 99%

Understanding the kinetic mechanism of RNA single base pair formation

Chen

2015

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

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RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/ dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model. RNAs perform critical cellular functions at the level of gene expression and regulation (1-4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7,8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9-19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19-36). However, due to the complexity of the RNA folding energy landscape (37-46) and the limitations of experimental tools (47-55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18,19,(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on-and offpathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, bas...

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confidence: 99%

Understanding the kinetic mechanism of RNA single base pair formation

Chen

2015

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

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“…Substitution of 2-AP for A has been shown previously to have a small influence on the stability of RNA (Zagórowska and Adamiak 1996;Jiao et al 2002;Liu et al 2008;Sarkar et al 2010). It was important to determine if the 2-AP/U and A/U base pair were similar in stability in the context of a hairpin stem.…”

Section: Discussionmentioning

confidence: 99%

“…The fluorescence of 2-AP is sensitive to its local environment (Menger et al 2000;Ballin et al 2007;Sarkar et al 2010;Rau et al 2012). The change in fluorescence with temperature has been used to examine the change in local structure as a molecule denatures.…”

Section: Modulation Of 2-ap Fluorescence By Thermal Denaturation Of Rmentioning

confidence: 99%

“…The change in fluorescence with temperature has been used to examine the change in local structure as a molecule denatures. Typically, when a basepaired 2-AP base denatures, the fluorescence intensity of the base increases due to the decrease in stacking of the base with its neighbors; conversely, an unpaired 2-AP base decreases its fluorescence as it denatures due to increased interactions with its neighbors in the single-stranded form (Menger et al 2000;Jiao et al 2002;Hardman and Thompson 2006;Ballin et al 2007;Sarkar et al 2009Sarkar et al , 2010Rau et al 2012;). The major goal of this study was to use the change in 2-AP fluorescence to investigate the structural ambiguity of the group II single nucleotide bulge loops.…”

Section: Modulation Of 2-ap Fluorescence By Thermal Denaturation Of Rmentioning

confidence: 99%

“…In addition to local environment, the fluorescence of 2-AP is strongly influenced by temperature (Menger et al 2000;Ballin et al 2007;Sarkar et al 2010;Rau et al 2012). Therefore, to accurately use 2-AP fluorescence to monitor the structural changes, fluorescent measurements for the 2-AP labeled RNA hairpins were corrected for the fluorescence of a five nucleotide short strand of RNA that was analogous to the region of the RNA hairpin where the 2-AP was inserted (Ballin et al 2007).…”

Section: Modulation Of 2-ap Fluorescence By Thermal Denaturation Of Rmentioning

confidence: 99%

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Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

Dishler

McMichael

Serra

2015

RNA

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Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°3 7 , and T M for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5 ′ or 3 ′ position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5 ′ or 3 ′ side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.

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Sequence‐Dependent Structural Dynamics of Primate Adenosine‐to‐Inosine Editing Substrates

et al. 2012

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Humans have the highest level of adenosine-to-inosine (A-to-I) editing amongst primates, yet the reasons for this difference remain unclear. Sequence analysis of the Alu Sg elements (A-to-I RNA substrates) corresponding to the Nup50 gene in human, chimp, and rhesus reveals subtle sequence variations surrounding the edit sites. We have developed three constructs that represent human (HuAp5), chimp (ChAp5), and rhesus (RhAp5) Nup50 Alu Sg A-to-I editing substrates. Here, 2-aminopurine (2-Ap) was substituted for edited adenosine (A5) so as to monitor the fluorescence intensity with respect to temperature. UV and steady-state fluorescence (SSF) T(M) plots indicate that local and global unfolding are coincident, with the human construct displaying a T(M) of approximately 70°C, compared to 60°C for chimp and 54°C for rhesus. However, time-resolved fluorescence (TRF) resolves three different fluorescence lifetimes that we assign to folded, intermediate(s), and unfolded states. The TRF data fit well to a two-intermediate model, whereby both intermediates (M, J) are in equilibrium with each other, and the folded/unfolded states. Our model suggests that, at 37°C, human state J and the folded state will be the most heavily populated in comparison to the other primate constructs. In order for adenosine deaminase acting on RNA (ADAR) to efficiently dock, a stable duplex must be present that corresponds to the human construct, globally. Next, the enzyme must "flip out" the base of interest to facilitate the A-to-I conversion; a nucleotide in an intermediate-like position would enhance this conformational change. Our experiments demonstrate that subtle variations in RNA sequence might contribute to the high A-to-I editing levels found in humans.

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Loop and stem dynamics during RNA hairpin folding and unfolding

Cited by 36 publications

References 26 publications

Understanding the kinetic mechanism of RNA single base pair formation

Understanding the kinetic mechanism of RNA single base pair formation

Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

Sequence‐Dependent Structural Dynamics of Primate Adenosine‐to‐Inosine Editing Substrates

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