Cooperative RNA Folding under Cellular Conditions Arises from Both Tertiary Structure Stabilization and Secondary Structure Destabilization

RNA folding has been studied extensively in vitro, typically under dilute solution conditions and abiologically high salt concentrations of 1 M Na⁺ or 10 mM Mg²⁺. The cellular environment is very different, with 20-40% crowding and only 10-40 mM Na⁺, 140 mM K⁺, and 0.5-2.0 mM Mg²⁺. As such, RNA structures and functions can be radically altered under cellular conditions. We previously reported that tRNA^phe secondary and tertiary structures unfold together in a cooperative two-state fashion under crowded in vivo-like ionic conditions, but in a noncooperative multistate fashion under dilute in vitro ionic conditions unless in nonphysiologically high concentrations of Mg²⁺. The mechanistic basis behind these effects remains unclear, however. To address the mechanism that drives RNA folding cooperativity, we probe effects of cellular conditions on structures and stabilities of individual secondary structure fragments comprising the full-length RNA. We elucidate effects of a diverse set of crowders on tRNA secondary structural fragments and full-length tRNA at three levels: at the nucleotide level by temperature-dependent in-line probing, at the tertiary structure level by small-angle X-ray scattering, and at the global level by thermal denaturation. We conclude that cooperative RNA folding is induced by two overlapping mechanisms: increased stability and compaction of tertiary structure through effects of Mg²⁺, and decreased stability of certain secondary structure elements through the effects of molecular crowders. These findings reveal that despite having very different chemical makeups RNA and protein can both have weak secondary structures in vivo leading to cooperative folding.