Cooperative Tertiary Interaction Network Guides RNA Folding

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends on the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold. [Display omitted] ► Tertiary interactions form in native-like folding intermediates of a group I ribozyme ► RNA tertiary interactions become cooperatively linked in folding intermediates ► The network of cooperative interactions is remodeled in the native state ► Cooperativity depends on the native architecture of the ribozyme Most tertiary interactions are not required for cementing the final fold of a structured RNA. Rather, they function early in RNA folding, forming a cooperative network that suppresses the formation of nonnative structures and guides the search for the unique, stable fold of the native state.