Perturbation of the Hierarchical Folding of a Large RNA by the Destabilization of its Scaffoldʹs Tertiary Structure

The P4–P6 domain serves as a scaffold against which the periphery and catalytic core organize and fold during Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. The most prominent structural motif of the P4–P6 domain is the tetraloop–tetraloop receptor interaction which “clamps” the distal parts of its hairpin-like structure. Destabilization of the tertiary structure of the P4–P6 domain by perturbation of the tetraloop–tetraloop receptor interaction alters the Mg2+-mediated folding pathway. The folding hierarchy of P5c≈P4-P6>periphery>catalytic core that is a striking attribute of the folding of the wild-type RNA is abolished. The initial steps in folding of the mutant RNA are ≥50-fold faster than those of the wild-type ribozyme with the earliest observed tertiary contacts forming around regions known to specifically bind Mg2+. The interaction between the mutant tetraloop and the tetraloop receptor appears coincidently with slowly forming catalytic core tertiary contacts. Thus, the stability conferred upon the P4–P6 domain by the tetraloop–tetraloop receptor interaction dictates the preferred folding pathway by stabilizing an early intermediate. A sub-denaturing concentration of urea diminishes the early barrier to folding the wild-type ribozyme along with complex effects on the subsequent steps of folding the wild-type and mutant RNA.