Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme

Abstract

Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group 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 interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (E ∆P5abc ). Equilibrium gel shift experiments show that the affinity of the P5abc and E ∆P5abc RNAs is exceptionally strong, with a K d of ∼100 pM at 10 mM MgCl 2 (at 37°C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility 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 indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.Large RNA molecules, like proteins, readily form specific molecular shapes adapted for ligand binding and catalysis. Catalytic RNAs including group I and group II introns and RNase P have compact interiors, involving close association of several segments of secondary structure. Close packing of RNA helices requires extensive screening of the phosphodiester backbone by cations in solution (1, 2). Furthermore, noncanonical base pairs and hairpin loops provide distinct arrays of hydrogen bond donors and acceptors and irregular surface features that serve as recognition sites for RNA helices in the formation of higher order structures (3)(4)(5)(6)(7)(8). How tertiary contacts, charge screening, and other factors stabilize RNA helix packing is not well understood.The X-ray crystal structure of the P4-P6 domain from the Tetrahymena thermophila group I intron provided the first detailed view of an extended tertiary interface in RNA (9). In the 160 nucleotide domain, a flexible internal loop, J5/5a, allows the backbone to make a ∼180°bend, enabling parallel association of two coaxially stacked helical segments (Figure 1). One-half of the P4-P6 domain, a three-helix subdomain called P5abc, binds at two distinct locations to the other half of the domain, constituted by the coaxially stacked P5, P4, and P6 helices (Figure 1). The A-rich bulge of P5abc contacts the minor groove of helix P4 by backbone and base-mediated hydrogen bonds. Additionally, a GAAA tetraloop in P5abc docks into its tetraloop receptor by both cross-helical base stacking and extensive hydrogen bonding. Similar GAAA tetraloop/tetraloop receptor interactions are thought to occur in other large RNAs including group I...

Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme

“…In addition to being the first subdomain to form, P5abc is the most stable part of the ribozyme, remaining ordered at the lowest Mg 2+ concentration and the highest temperature (19,20). Consistent with the view of P5abc as a critical part of the structure, its deletion gives a ribozyme (E ΔP5abc ) that is substantially destabilized and has greatly diminished catalytic activity (4,21), defects that are rescued by addition of P5abc as a separate molecule (4,22). Because of its stability and rapid formation of tertiary structure, P5abc was proposed to facilitate folding by nucleating formation of the P4-P6 domain, which would then provide the scaffold for the entire ribozyme (3,23).…”

“…S5 in Supporting Information, and data not shown). This may be because the protections and enhancements are relatively weak for the E ΔP5abc ribozyme [ (21,33) and Fig. S4 in Supporting Information], perhaps reflecting a dynamic or 'floppy' structure, which may also underlie the result that the slowest folding steps were not detected in time-resolved footprinting experiments.…”

New pathways in folding of the Tetrahymena group I RNA enzyme

“…Further, these results raise the possibility that there is an additional folding pathway for the wild-type ribozyme in which P5abc acquires tertiary structure last rather than ®rst ( Figure 5(a)). However, although the global folds are similar (Doherty et al, 1999), the active conformation attained by E ÁP5abc is not the native conformation reached by the wild-type, as the folded E ÁP5abc continues to lack P5abc and is compromised in activity (Joyce et al, 1989;M. Engelhardt et al, unpublished results).…”

Direct Measurement of Tertiary Contact Cooperativity in RNA Folding

“…Surprisingly, the number of RNA systems in which cooperativity has been rigorously dissected is limited. 19,20 Indeed, quantitative energetic dissection of the cooperativity underlying formation of RNA tertiary structure has remained difficult for experimental and conceptual SmFRET experiments avoid this pitfall because the high accuracy achievable in measurement of equilibria well away from a folding midpoint 15,22 allows direct comparison of equilbria under identical conditions. For example, in bulk, a 5% folding signal may be difficult to distinguish from, for example, a 3% signal; in the corresponding smFRET experiment, the folding signal is 100% (for 5% of the time), making determination of the amount of folded molecule present straightforward.…”