Hinge Stiffness Is a Barrier to RNA Folding

Schlatterer, Jörg C.; Kwok, Lisa W.; Lamb, Jessica S.; Park, Hye Yoon; Andresen, Kurt; Brenowitz, Michael; Pollack, Lois

doi:10.1016/j.jmb.2008.04.013

Cited by 48 publications

(72 citation statements)

References 36 publications

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“…Overall, the P4-P6 kinetic and thermodynamic constants obtained by smFRET measurements agree well with those obtained by multiple bulk approaches, including hydroxyl radical footprinting, stopped-flow fluorescence with a pyrene label, and small angle x-ray scattering (41)(42)(43)(44). Nevertheless, although some of these approaches provide additional structural information, none can match smFRET for the range of conditions that can be investigated, and the ease at which reversible folding can be followed.…”

supporting

confidence: 66%

“…1, A and B). P4-P6 was the first RNA with a side-by-side arrangement of helices to have its structure elucidated by x-ray crystallography and has been the subject of numerous folding studies (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The knowledge provided by these prior studies along with its relative simplicity, with only two long-range tertiary interactions, render P4-P6 an attractive target for addressing the general issue of complexity in RNA folding.…”

mentioning

confidence: 99%

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Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA

Greenfeld

Solomatin

Herschlag

2011

Journal of Biological Chemistry

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RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a nonconformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave nearuniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior.Highly structured RNAs such as the ribosome, spliceosome, and riboswitches must fold to function and undergo conformational changes in the course of their function (1, 2). With the majority of the transcriptome largely unexplored, it seems likely that additional roles of RNA structure will emerge (3). Yet the folding and conformational changes of structured RNAs remain poorly understood.Two general models for how RNAs fold have been widely discussed. In one view, RNA folding is a simple process, particularly in comparison to protein folding (4, 5). In a contrasting view, RNA folding is complex, replete with numerous deep kinetic traps on a rugged energy landscape (6, 7). The simple view is derived from the limited diversity of RNA structural components and the hierarchical nature of RNA folding. The isolated RNA secondary structure is highly stable in contrast to the marginal stability of isolated protein ␣-helices or ␤-sheets. In transitioning from a secondary to a tertiary structure, junctions that link RNA helices can favor particular orientations of the helical elements that are then enforced by modular tertiary motifs (8, 9). In contrast, globular proteins appear to have more extensive and interconnected packing arrangements with less distinct separation in the formation of the se...

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supporting

confidence: 66%

mentioning

confidence: 99%

Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA

Greenfeld

Solomatin

Herschlag

2011

Journal of Biological Chemistry

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“…The folding of P4-P6 has been followed in multiple studies, with folding rate constants of 2-30 s −1 observed across a range of conditions (52)(53)(54)(55)(61)(62)(63)(64)(65). However, interpretation of these overall rate constants has been stymied by a lack of knowledge of which individual folding steps were followed and rate limiting.…”

Section: Resultsmentioning

confidence: 99%

“…The control afforded by this well-behaved, established system provides an opportunity to deepen understanding and develop and test generalizable principles beyond this model system, provided that a detailed framework is in place to provide context for interpretation. Here, we use single-molecule FRET (smFRET) with a series of P4-P6 mutants to isolate and measure specific folding transitions, extending beyond measurements of overall folding rates (52)(53)(54)(55)(61)(62)(63)(64)(65) and constructing a kinetic and thermodynamic framework for P4-P6 RNA. This framework defines a preferred folding pathway, allows this pathway and the partitioning of states to be interpreted in structural terms, helps to uncover the origin of the effects of mutations on folding rates and pathways, and suggests a generalized framework for considering RNA tertiary folding.…”

mentioning

confidence: 99%

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Bisaria

Greenfeld

Limouse

et al. 2016

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

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The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.RNA folding | single-molecule FRET | kinetics | folding pathways | RNA tertiary motifs S tructured RNAs play central roles in biology, in the splicing and alternative splicing of eukaryotic pre-mRNAs, in the synthesis of proteins and their transport, and in chromosome maintenance (1-4). Beyond the RNAs and RNA-protein machines involved in these processes, it has been increasingly recognized that Nature has taken extensive advantage of RNA at multiple levels of gene regulation, and considerable current efforts are focused on uncovering the pathways and molecular mechanisms that underlie the functions of small RNAs and long noncoding RNAs (2, 5-8). The pervasive functions of RNA in biology underscore the importance of understanding RNA's fundamental physical properties and, ultimately, of using this understanding to predict the kinetics and thermodynamics of folding and conformational transitions responsible for RNA function.Decades of characterization of RNA folding and structure have led to generalizable principles and provided biological insights. The observations that RNA encodes genetic information, forms highly stable local structures, and can catalyze reactions provided support for the possibility that early life evolved using functional RNAs (9-12). This high stability was also re...

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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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Hinge Stiffness Is a Barrier to RNA Folding

Cited by 48 publications

References 36 publications

Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA

Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Understanding the kinetic mechanism of RNA single base pair formation

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