The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc-deletion variant (E ΔP5abc ) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E ΔP5abc forms its earliest stable tertiary structure on the millisecond time scale, ∼5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E ΔP5abc in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E ΔP5abc ribozyme is much slower (∼0.6 min −1 ), similar to the wild type. Also similar, only a small fraction of E ΔP5abc attains the native state on this timescale under standard conditions at 25 °C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wildtype ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E ΔP5abc refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.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 n...