title: 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.
authors: Department of Biochemistry, Stanford University, Stanford, CA 94305;
authors: Namita
authors: N
authors: Bisaria
authors: Department of Biochemistry, Stanford University, Stanford, CA 94305; Department of Chemical Engineering, Stanford University, Stanford, CA 94305;
authors: Max
authors: M
authors: Greenfeld
authors: Department of Applied Physics, Stanford University, Stanford, CA 94305;
authors: Charles
authors: C
authors: Limouse
authors: Department of Applied Physics, Stanford University, Stanford, CA 94305;
authors: Dmitri S
authors: DS
authors: Pavlichin
authors: Department of Applied Physics, Stanford University, Stanford, CA 94305;
authors: Hideo
authors: H
authors: Mabuchi
authors: Department of Biochemistry, Stanford University, Stanford, CA 94305; Department of Chemical Engineering, Stanford University, Stanford, CA 94305; Department of Chemistry, Stanford University, Stanford, CA 94305 herschla@stanford.edu.
authors: Daniel
authors: D
authors: Herschlag
mesh_terms: D020029:Base Pairing; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D059372:Nucleotide Motifs; D017354:Point Mutation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D059370:RNA Folding; D000072760:Single Molecule Imaging / Q000379:methods; D013816:Thermodynamics
chemical_list: D012313:RNA
keywords: RNA folding; RNA tertiary motifs; folding pathways; kinetics; single-molecule FRET