The Role of Topological Constraints in RNA Tertiary Folding and Dynamics.

Abstract

Functional RNA molecules must fold into highly complex three-dimensional (3D) structures and undergo precise structural dynamics in order to carry out their biological functions. However, the principles that govern RNA 3D folding and dynamics remain poorly understood. Recent studies have proposed that topological constraints arising from the basic connectivity and steric properties of RNA secondary structure strongly confine the 3D conformation of RNA junctions and thus may contribute to the specificity of RNA 3D folding and dynamics. Herein, this hypothesis is explored in quantitative detail using a combination of computational heuristic models and the specially developed coarse-grained molecular dynamics model TOPRNA. First, studies of two-way junctions provide new insight into the significance and mechanism of action of topological constraints. It is demonstrated that topological constraints explain the directionality and amplitude of bulge-induced bends, and that long-range tertiary interactions can modify topological constraints by disrupting non-canonical pairing in internal loops. Furthermore, topological constraints are shown to define free energy landscapes that coincide with the distribution of bulge conformations in structural databases and reproduce solution NMR measurements made on bulges. Next, TOPRNA is used to investigate the contributions of topological constraints to tRNA folding and dynamics. Topological constraints strongly constrain tRNA 3D conformation and notably discriminate against formation of non-native tertiary contacts, providing a sequence-independent source of folding specificity. Furthermore, topological constraints are observed to give rise to thermodynamic cooperativity between distinct tRNA tertiary interactions and encode functionally important 3D dynamics. Mutant tRNAs with unnatural secondary structures are shown to lack these favorable characteristics, suggesting that topological constraints underlie the evolutionary conservation of tRNA secondary structure. Additional studies of a non-canonical mitochondrial tRNA show that increased topological constraints can reduce the entropic cost of tertiary folding, and that disruptions of topological constraints explain the pathogenicity of a insertion mutation in this tRNA. UV melting experiments verify these findings. Finally, TOPRNA is used to study the topological constraints of the 197 nucleotide Azoarcus Group I ribozyme. It is shown that topological constraints strongly confine this RNA and provide a mechanism for encoding tertiary structure specificity and cooperative hierarchical folding behavior.