The dynamics and equilibria of RNA conformational changes are critical features of the multitude of RNA-mediated reactions that take place in living cells. RNA enzymes or ribozymes have provided the gold standard for RNA structure-function analyses for the past two decades because they reveal their assembly into functional structures through catalytic activity. But, despite two decades of research our understanding of the basic mechanisms of RNA assembly remains limited. The hairpin ribozyme, encoded in the (-)TRSV satellite RNA, is a small catalytic motif that catalyzes sequence-specific reversible self-cleavage. Existing structural, biochemical, and kinetic data for the hairpin ribozyme, and the availability of a yeast-based in vivo reporter system make this motif and ideal model for studying intracellular RNA assembly pathways. This system will be used to determine the kinetic and thermodynamic influences on ribozyme assembly during transcription and translation inside living cells, and to monitor whether RNAs interact within discrete folding domains during co-transcriptional ribozyme assembly. Ultimately this work will improve our understanding of the basic principles governing RNA assembly in vivo.
