Kinetics and thermodynamics make different contributions to RNA folding in vitro and in yeast.

RNAs somehow adopt specific functional structures despite the capacity to form alternative nonfunctional structures with similar stabilities. We analyzed RNA assembly during transcription in vitro and in yeast using hairpin ribozyme self-cleavage to assess partitioning between functional ribozyme structures and nonfunctional stem loops. Complementary insertions located upstream of the ribozyme inhibited ribozyme assembly more than downstream insertions during transcription in vitro, consistent with a sequential folding model in which the outcome is determined by the structure that forms first.