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

Abstract

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...

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

“…This ability, along with homogenous molecular behavior (ref. 90 and Materials and Methods), allows construction of a folding framework under uniform conditions, avoiding uncertainties and complications from extrapolation of RNA behavior across conditions (62,82,91). Example traces and data quality analyses for all constructs can be found in Dataset S1.…”

“…1C) (60). Also, prior P4-P6 smFRET work revealed molecular heterogeneity from covalent damage caused by standard RNA treatments and showed that this damage could be avoided to yield highly homogeneous populations (90).…”

“…The smP4-P6 variants were prepared by a five-piece splinted ligation with protocols slightly altered from those described previously (60,90,117). Prior studies revealed that standard laboratory protocols introduce covalent differences in the RNA and that these modifications can affect the folding of individual molecules (90).…”

Structural dynamics of a single-stranded RNA–helix junction using NMR

“…2C). These resonances are therefore unlikely to arise from UV chemical damage to the RNA (Greenfeld et al 2011;Kladwang et al 2012) or from N + 1 products during in vitro RNA transcription (Milligan et al 1987;Cazenave and Uhlenbeck 1994;Pleiss et al 1998;Helm et al 1999). Rather, these additional resonances could reflect an alternative conformation for adenine residues A25, A27, and A28 near the junction that is in slow exchange with the major state.…”

Theoretical Search for RNA Folding Nuclei

“…At the same time a three-helix junction (P5abc) within the P4-P6 domain folds at least 25 times more rapidly in isolation than when being a part of the wild-type P4-P6 RNA [51]. The fastest observed rate constants for P4-P6 folding are on the order of 10 s −1 [52], much slower than the formation of basic structures such as RNA hairpins, which form on the microsecond timescale [53,54].…”