Hidden Structural Modules in a Cooperative RNA Folding Transition

Large-scale, cooperative rearrangements underlie the functions of RNA in RNA-protein machines and gene regulation. To understand how such rearrangements are orchestrated, we used high-throughput chemical footprinting to dissect a seemingly concerted rearrangement in P5abc RNA, a paradigm of RNA folding studies. With mutations that systematically disrupt or restore putative structural elements, we found that this transition reflects local folding of structural modules, with modest and incremental cooperativity that results in concerted behavior. First, two distant secondary structure changes are coupled through a bridging three-way junction and Mg²⁺-dependent tertiary structure. Second, long-range contacts are formed between modules, resulting in additional cooperativity. Given the sparseness of RNA tertiary contacts after secondary structure formation, we expect that modular folding and incremental cooperativity are generally important for specifying functional structures while also providing productive kinetic paths to these structures. Additionally, we expect our approach to be useful for uncovering modularity in other complex RNAs.