Understanding the Role of Three-Dimensional Topology in Determining the Folding Intermediates of Group I Introns

Many RNA molecules exert their biological function only after folding to unique three-dimensional structures. For long, noncoding RNA molecules, the complexity of finding the native topology can be a major impediment to correct folding to the biologically active structure. An RNA molecule may fold to a near-native structure but not be able to continue to the correct structure due to a topological barrier such as crossed strands or incorrectly stacked helices. Achieving the native conformation thus requires unfolding and refolding, resulting in a long-lived intermediate. We investigate the role of topology in the folding of two phylogenetically related catalytic group I introns, the Twort and Azoarcus group I ribozymes. The kinetic models describing the Mg2+-mediated folding of these ribozymes were previously determined by time-resolved hydroxyl (⋅OH) radical footprinting. Two intermediates formed by parallel intermediates were resolved for each RNA. These data and analytical ultracentrifugation compaction analyses are used herein to constrain coarse-grained models of these folding intermediates as we investigate the role of nonnative topology in dictating the lifetime of the intermediates. Starting from an ensemble of unfolded conformations, we folded the RNA molecules by progressively adding native constraints to subdomains of the RNA defined by the ⋅OH time-progress curves to simulate folding through the different kinetic pathways. We find that nonnative topologies (arrangement of helices) occur frequently in the folding simulations despite using only native constraints to drive the reaction, and that the initial conformation, rather than the folding pathway, is the major determinant of whether the RNA adopts nonnative topology during folding. From these analyses we conclude that biases in the initial conformation likely determine the relative flux through parallel RNA folding pathways

Understanding the Role of Three-Dimensional Topology in Determining the Folding Intermediates of Group I Introns

Many RNA molecules exert their biological function only after folding to unique three-dimensional structures. For long, noncoding RNA molecules, the complexity of finding the native topology can be a major impediment to correct folding to the biologically active structure. An RNA molecule may fold to a near-native structure but not be able to continue to the correct structure due to a topological barrier such as crossed strands or incorrectly stacked helices. Achieving the native conformation thus requires unfolding and refolding, resulting in a long-lived intermediate. We investigate the role of topology in the folding of two phylogenetically related catalytic group I introns, the Twort and Azoarcus group I ribozymes. The kinetic models describing the Mg2+-mediated folding of these ribozymes were previously determined by time-resolved hydroxyl (⋅OH) radical footprinting. Two intermediates formed by parallel intermediates were resolved for each RNA. These data and analytical ultracentrifugation compaction analyses are used herein to constrain coarse-grained models of these folding intermediates as we investigate the role of nonnative topology in dictating the lifetime of the intermediates. Starting from an ensemble of unfolded conformations, we folded the RNA molecules by progressively adding native constraints to subdomains of the RNA defined by the ⋅OH time-progress curves to simulate folding through the different kinetic pathways. We find that nonnative topologies (arrangement of helices) occur frequently in the folding simulations despite using only native constraints to drive the reaction, and that the initial conformation, rather than the folding pathway, is the major determinant of whether the RNA adopts nonnative topology during folding. From these analyses we conclude that biases in the initial conformation likely determine the relative flux through parallel RNA folding pathways

() Dose–response determinations of the ·OH cleavage of DNA (black) and RNA (blue) following exposure of the nucleic acids to 5 mM Fe(II)-EDTA and 44 mM HO for the indicated period of time

<p><b>Copyright information:</b></p><p>Taken from "Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins"</p><p>Nucleic Acids Research 2006;34(6):e48-e48.</p><p>Published online 31 Mar 2006</p><p>PMCID:PMC1421499.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The data are best-fit by single exponentials with amplitudes and half-times of A = 0.54 & 0.44, A = 0.31 ± 0.02 & 0.30 ± 0.02 and t = 4.2 ± 0.9 and 6.1 ± 1.1 ms for DNA and RNA, respectively. The DNA dose–response curve was determined by quantification of the autoradiogram shown in . () The extent of ·OH mediated cleavage of DNA (black) and RNA (blue) determined at 44 mM HO with a 5 ms reaction time as a function of Fe(II)-EDTA concentration. The solid lines are fits of the data to a simple saturation function. Open and closed symbols are replicate experiments in both (A) and (B)

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

ZBP1 is a member of a family of RNA-binding proteins that regulate RNA localization, stability, and translation of specific target mRNAs. Identifying target mRNAs for ZBP1 family members based solely on mRNA sequence has been difficult. Here, Chao and colleagues use ZBP1 recognition of the β-actin zipcode RNA to identify additional RNA targets by combining the characterization of the exact cis-acting sequence elements with the structural restraints of ZBP1's mode of RNA binding. The biological relevance of the predicted mRNA targets of ZBP1 is established by showing that the mRNA of a protein found in neurons is mislocalized in ZBP1 knockout neurons. Thus, this study demonstrates a novel method for identifying the RNA targets of RNA-binding proteins