Tertiary Interactions Maintain the Balance of Stability, Folding Efficiency and Speed in a Large Catalytic Bacterial RNA

Abstract

Like their protein counterparts, many non-coding RNAs, such as ribozymes, must fold into a unique 3D structure suited for their biological actions. These RNA structures are stabilized by an array of conserved tertiary interactions between residues far apart in sequence. However, the extent of thermodynamic coupling between tertiary interactions in RNA is little understood. To probe the basis for cooperative folding in RNA, we perturbed the folding energetics of a bacterial group I ribozyme with mutations that disrupt five conserved tertiary interaction sites. The assembly of core helices into compact native-like intermediates was measured by SAXS and native PAGE. Formation of the native state was probed with activity assays and hydroxyl radical footprinting of the RNA backbone. Although all of the mutants were catalytically active, their folding landscapes were significantly changed. Single mutations destabilized the compact intermediates and disrupted the thermodynamic link between tertiary interactions measured through double mutant cycles. Native PAGE and footprinting showed that loss of a single tertiary interaction affected the stability of other structural domains due to competition with non-native structures. Stopped-flow SAXS and hydroxyl radical footprinting with millisecond resolution showed that disruption of tertiary interactions does not hamper the rapid initial collapse of the RNA chain, but changes the folding hierarchy of the structural domains and the structures of populated intermediates. These results shed light on the interplay of the conserved tertiary interactions and electrostatic forces in the folding of large RNA molecules and delineate the mechanisms by which evolved RNA sequences determine the outcome of an otherwise nonspecific counterion-mediated collapse.