Deletion of the P5abc peripheral element accelerates early and late folding steps of the Tetrahymena group I ribozyme.

Adoption of a specific three-dimensional structure for RNA is a complex process that typically involves the formation and accumulation of intermediates. Most fundamentally, this behavior arises because local RNA structure can form rapidly and can be stable in the absence of enforcing long-range or global structure, allowing partially structured intermediates to form and accumulate. The stability of RNA structure may lead, in general, to a hierarchy in its folding process, with global tertiary structure forming primarily from pre-formed units of secondary and local tertiary structure (5). This folding behavior contrasts with that of many proteins, whose local elements of secondary and tertiary structure are unstable in the absence of the global fold of a subunit or domain and are therefore formed in concert (6, 7). This hierarchy may facilitate the folding of RNAs, as stable structure that forms early can provide a scaffold upon which additional structure is built. However, hierarchical folding also introduces the possibility that incorrect structure will form and be stable, generating misfolded or ‘kinetically trapped’ intermediates, which require partial or complete unfolding to continue folding productively. Indeed, RNA misfolding has been recognized since early studies of tRNA (8, 9) and has more recently been shown to be a near-ubiquitous feature of folding for large, multi-domain RNAs (10-16). A wealth of information on folding steps and intermediates has been obtained from studies of the Tetrahymena group I ribozyme. The ribozyme includes a core and several peripheral elements that surround the core. Several lines of evidence have indicated that one of the peripheral elements, termed P5abc, plays a vital role in folding. Time-resolved oligonucleotide hybridization and footprinting studies showed that tertiary folding begins with P5abc, followed closely by the rest of the P4-P6 domain (2, 3, 17, 18) (Fig. 1). The rest of the ribozyme then acquires stable structure through a series of intermediates (2, 3, 18), while P5abc and the entire P4-P6 domain retain structure in each subsequent intermediate. In addition to being the first subdomain to form, P5abc is the most stable part of the ribozyme, remaining ordered at the lowest Mg2+ 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 (EΔP5abc) 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). Figure 1 Tertiary folding of the Tetrahymena ribozyme. Double-stranded regions that do not have stable tertiary structure are shown as gray cylinders, and regions that have formed tertiary structure are colored as follows. The core domains, P4-P6 and P3-P8, are ... On the other hand, studies of folding kinetics called into question the productive role of early P5abc folding. Several mutations that weaken native structure within P5abc increase the rate of stable tertiary structure formation within the P3-P8 domain, suggesting that native structure formed early within P5abc is transiently disrupted at a later stage of folding (14, 24, 25), and similar mutations accelerate refolding of a long-lived misfolded intermediate that is formed by most of the ribozyme population during folding [(26); see also (15, 27, 28)]. Further, the small fraction of the ribozyme that avoids the misfolded conformation folds only slightly more slowly upon deletion of P5abc (15). These studies indicated that under the conditions of these experiments (10 mM Mg2+ and <20 mM Na+, ‘low salt’ conditions), the early formation of P5abc provides no overall benefit and even appears to slow folding steps involving the ribozyme core. However, it remained possible that P5abc accelerated early folding steps, but the beneficial effect was not detected because it was obscured by later steps that were unaffected or slowed by P5abc. More recently, it has been shown that mutation of a tertiary contact within P5abc or addition of the denaturant urea to the wild-type ribozyme accelerates even early folding steps, most simply suggesting that structure within P5abc is detrimental even early in folding (29). To more fully understand the role of P5abc structure in folding, here we use footprinting and activity assays to systematically examine early and late folding steps, comparing folding of the wild-type ribozyme and the EΔP5abc variant under ‘low salt’ solution conditions. Strikingly, we find that deletion of P5abc accelerates even the early folding steps, suggesting that, under these conditions, most or all of the partially structured intermediates that accumulate are kinetically trapped. Further, although P5abc has little effect on the rate constant for native state formation along the sparsely populated pathway that avoids the long-lived misfolded conformation, it dramatically slows refolding of the heavily populated misfolded intermediate. Finally, we build on previous studies by showing that P5abc stabilizes the native state relative to partially structured and unstructured intermediates, supporting previous results in providing a rationale for its presence in the natural RNA even if the deleterious effects observed here are also present during folding in vivo.