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

Abstract

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...

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

“…RNA tertiary formation, including that for formation of the TL/ TLR studied herein, is much slower than expected for diffusional collision; thus, there must be an additional step or steps preceding formation of the contact (21,27,48,49). Although in principle there might only be a need for two steps, tertiary contact rearrangement and alignment (diffusional collision), our results provide evidence for an additional step.…”

“…2B). Prior work demonstrated equivalent P4-P6 folding in Mg 2+ and Ba 2+ upon ablation of the MC, and independent studies demonstrated equal ion atmosphere occupancy for Mg 2+ and Ba 2+ surrounding a nucleic acid (21,45). Thus, we could change the electrostatic environment without complications from site-specific metal ion binding and formation of an additional tertiary contact ( (21) concentrations (Fig.…”

“…P4-P6 RNA has been widely studied as a model for RNA tertiary folding (e.g., refs. [35][36][37][38][39], and our recent thermodynamic and kinetic framework for its folding provides the necessary structural, thermodynamic, and kinetic context for interpretation of the results herein (21). P4-P6 has two tertiary contacts, a canonical GAAA-11nt TL/TLR and a metal core/metal core receptor (MC/MCR), and their formation bring its two helical stacks into a roughly parallel conformation (37,(40)(41)(42)(43) (Fig.…”

“…Alternatively, differing connections between tertiary elements could distort the motif's structure, alter the interactions made, and consequently lead to energetic interdependence between the motif and RNA elements that span the contacts. Initial tests of the kinetic behavior of one tertiary contact motif, the tetraloop/tetraloop receptor (TL/TLR), embedded within different structures revealed a common unfolding rate constant in different contexts, providing evidence for a modular, locally defined physical process (21).…”

“…The thermodynamic modularity of the tertiary folding energetics introduces the possibility that there may also be a conservation of the underlying properties that determines the kinetics of tertiary contact formation and dissolution. We can make kinetic predictions based on the diffusion-collision model for RNA (21), whereby folding rate constants are determined by the rate constant at which tertiary elements collide weighted by the probability that the tertiary elements are in a state competent to proceed to form the interactions of the stable contact. To uncover predictions from this model, we break the overall folding process down in this model into three steps (Fig.…”