Tertiary Interactions Determine the Accuracy of RNA Folding

Abstract

RNAs must fold into unique three-dimensional structures to function in the cell, but how each polynucleotide finds its native structure is not understood. To investigate whether the stability of the tertiary structure determines the speed and accuracy of RNA folding, docking of a tetraloop with its receptor in a bacterial group I ribozyme was perturbed by site-directed mutagenesis. Disruption of the tetraloop or its receptor destabilizes tertiary interactions throughout the ribozyme by 2-3 kcal/ mol, demonstrating that tertiary interactions form cooperatively in the transition from a native-like intermediate to the native state. Nondenaturing PAGE and RNase T1 digestion showed that base pairs form less homogeneously in the mutant RNAs during the transition from the unfolded state to the intermediate. Thus, tertiary interactions between helices bias the ensemble of secondary structures toward native-like conformations. Time-resolved hydroxyl radical footprinting showed that the wild type ribozyme folds completely within 5-20 ms. By contrast, only 40-60% of a tetraloop mutant ribozyme folds in 30-40 ms, with the remainder folding in 30 -200 s via non-native intermediates. Therefore, destabilization of tetraloop-receptor docking introduces an alternate folding pathway in the otherwise smooth energy landscape of the wild type ribozyme. Our results show that stable tertiary structure increases the flux through folding pathways that lead directly and rapidly to the native structure.

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

“…5 H and I), so these loops can be significant in determining folding cooperativity and pathways in some cases, although secondary structure base pairing is expected to be dominant. Although the generality of the finding that tertiary interactions can consolidate secondary structure formation is not clear, there are examples that suggest that this scenario can be realized in several RNA molecules (15,30,42).…”

On the importance of cotranscriptional RNA structure formation

“…One example is the cotranscriptional folding of the Tetrahymena ribozyme in vitro, which is twice as fast as the refolding of the fully synthesized and denatured molecule, but slower than the cotranscriptional folding in vivo (Heilmann-Miller and Woodson 2003a). Cotranscriptional folding pathways in vivo need not be unique (Jackson et al 2006), and tertiary interactions can determine which of several possible folding pathways is chosen (Chauhan and Woodson 2008). Factors such as transcription speed and flanking sequences can also influence which pathway dominates (Koduvayur and Woodson 2004).…”

“…Due to the methodological challenges of investigating RNA folding in vivo and in real time, we currently have only limited insight into folding pathways in vivo (Sclavi et al 1998;Heilmann-Miller and Woodson 2003a;Jackson et al 2006;Chauhan and Woodson 2008). Numerous recent in vitro experiments that replicate specific aspects of the complex in vivo environment and rapid progress regarding in vivo methodologies (Adilakshmi et al 2009;Alexander et al 2011) are likely to change this.…”

RNA structure prediction: Progress and perspective

“…[97] Some knowledge-based methods can predict the structures with no size limitation, but their requirements for descriptions of known structures and for hands-on interaction with expert user limit their wider application. For physics-based models, one possible way to treat large-size RNAs is to reduce the sampling conformational space.…”