Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme

Group I intron RNAs contain a core of highly conserved helices flanked by p eripheral domains that stabilize the core structure. In the Tetrahymena gro up I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary in teractions between these two parts of the domain create an extensive solven t-inaccessible interface. We have examined the formation and stability of t his tertiary interface by providing the P5abc segment in trans to a Tetrahy mena ribozyme construct that lacks P5abc (E-Delta P5abc). Equilibrium gel s hift experiments show that the affinity of the P5abc and E-Delta P5abc RNAs is exceptionally strong, with a K-d of similar to 100 PM at 10 mM MgCl2 (a t 37 degrees C). Chemical and enzymatic footprinting shows that the RNAs ar e substantially folded prior to assembly of the complex. Solvent accessibil ity mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through i ndirect effects of the tertiary interface formation. This two-component sys tem facilitates quantitative examination of individual tertiary contacts th at stabilize the folded intron.