Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

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Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔG align , the probability of aligning tertiary contact partners, and ΔG tert , the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔG HJH ) or from changes in the electrostatic environment (ΔG +/− ) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔG tert ). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔG tert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.RNA folding | single-molecule FRET | RNA tertiary structure | folding kinetics | folding thermodynamics S tructured RNAs are integral to many biological processes, including translation, genome maintenance, and the regulation of gene expression (1, 2). These processes require RNA to fold into intricate 3D structures and to undergo a series of structural transitions (3, 4). As such, an important goal has been to characterize these states, in terms of their conformations and the rates and equilibria that describe the transitions between them (5-8). A more distant but ultimately far-reaching challenge is to quantitatively predict the kinetics and thermodynamics of RNA transitions, an accomplishment that would demonstrate fundamental understanding and effectuate engineering of these systems.Quantitative analyses of the melting temperatures for nucleic acid duplexes led to predictive rules for RNA secondary structure stability, known as nearest neighbor or Turner rules, such that the energetics of helix formation can be predicted by considering only the identity of each base pair, the identity of its immediate neighbors, and the salt concentration (9-11). In this model, the energetic contribution of each base pair step is additive and can be summed to predict the free energy to a...

“…1A). Once aligned, particular tertiary partners can interact and thereby provide stabilization of intermediate and folded states (25)(26)(27)30).…”

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Bisaria

Greenfeld

Limouse

et al. 2017

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“…RNA secondary is preformed and stable, and RNA tertiary contact partners come together due to diffusion of the RNA helices they are embedded in (66,104,109). RNA tertiary contact formation also typically requires local unfavorable conformational rearrangements (104,(110)(111)(112)(113)(114)(115) such that the rates of formation of these interactions are orders of magnitude slower than diffusional collision (104,116).…”

Section: Discussionmentioning

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

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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...

“…For example, junctions (linkers) limit the conformations of unfolded states (52)(53)(54) and can be rigidified by Mg 2þ . In support of this, the [Mg 2þ ]-dependent decrease in the entropic cost of TL-R docking is much more pronounced in the A 7 vs. U 7 constructs (Tables 1 and 2)-suggesting that [Mg 2þ ]-dependent rigidification of the linker is of large entropic benefit to the docking process.…”

Section: Discussionmentioning

confidence: 99%

Entropic origin of Mg ²⁺ -facilitated RNA folding

Fiore

Holmstrom

Nesbitt

2012

158

Mg 2þ is essential for the proper folding and function of RNA, though the effect of Mg 2þ concentration on the free energy, enthalpy, and entropy landscapes of RNA folding is unknown. This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop−receptor tertiary interaction as a function of [Mg 2þ ]. These measurements yield the barrier and standard state enthalpies, entropies, and free energies for an RNA tertiary transition, in particular, revealing the thermodynamic origin of [Mg 2þ ]-facilitated folding. Surprisingly, these studies reveal that increasing [Mg 2þ ] promotes tetraloop-receptor interaction by reducing the entropic barrier (−T ΔS ‡ dock ) and the overall entropic penalty (−T ΔS°d ock ) for docking, with essentially negligible effects on both the activation enthalpy (ΔH ‡ dock ) and overall exothermicity (ΔH°d ock ). These observations contrast with the conventional notion that increasing [Mg 2þ ] facilitates folding by minimizing electrostatic repulsion of opposing RNA helices, which would incorrectly predict a decrease in ΔH ‡ dock and ΔH°d ock with [Mg 2þ ]. Instead we propose that higher [Mg 2þ ] can aid RNA folding by decreasing the entropic penalty of counterion uptake and by reducing disorder of the unfolded conformational ensemble.T he folding of RNA proceeds hierarchically, whereby secondary structure is formed rapidly and subsequent slow helical packing is mediated by tertiary interactions (1, 2). RNA secondary structure prediction from the known thermodynamics is quite reliable (3), though correspondingly accurate prediction of tertiary structure remains a major challenge (1). Static tertiary structure data alone are also not enough to predict RNA functionality, as time-dependent conformational dynamics occur during biochemical processes (4, 5). As a result, one needs the full free energy, enthalpy, and entropy landscapes for folding. A major road block in achieving a predictive understanding of RNA folding landscapes is that they are often "rugged,", i.e., with alternative conformations acting as kinetic traps (6, 7). Moreover, the electrostatic challenge of folding a charged biopolymer highlights the particularly critical role of Mg 2þ and other counterions in the folding process.Characterization of folding transition states-and the role of Mg 2þ in stabilizing transition states-remains a crucial bottleneck for reconciling the kinetics and thermodynamics of RNA folding (8-13). Some insight into the free energy landscapes for RNA folding can be obtained from temperature-dependent stopped-flow kinetic studies, which offer the ability to deconstruct free energy barriers (ΔG ‡ ) into enthalpic (ΔH ‡ ) and entropic (−TΔS ‡ ) components. However, with such methods, only the net rate constant (i.e., k total ¼ k fold þ k unfold ) for approach to equilibrium can be observed, which requires strong assumptions (e.g., that k fold ≫ k unfol...