Influence of substrate structure on in vitro ribozyme activity of a group II intron

Substrate sequences surrounding catalytic RNAs but not involved in specific, conserved interactions can severely interfere with in vitro ribozyme activity. Using model group II intron precursor transcripts with truncated or randomized exon sequences, we show that unspecific sequences within the 5' exon are particularly prone to inhibit both the forward and the reverse first splicing step (branching). Using in vitro selection, we selected efficient 5' exons for the reverse branching reaction. Precursor RNAs carrying these selected 5' exons reacted more homogeneously and faster than usual model precursor transcripts. This suggests that unfavorable structures induced by the 5' exon can introduce a folding step that limits the rate of in vitro self-splicing. These results stress how critical is the choice of the sequences retained or discarded when isolating folding domains from their natural sequence environments. Moreover, they suggest that exon sequences not involved in specific interactions are more evolutionarily constrained with respect to splicing than previously envisioned.