INFLUENCE OF SUBSTRATE STRUCTURE ON IN-VITRO RIBOZYME ACTIVITY OF A GROUP-II INTRON

Substrate sequences surrounding catalytic RNAs but not involved in spe cific, conserved interactions can severely interfere with in vitro rib ozyme activity. Using model group II intron precursor transcripts with truncated or randomized exon sequences, we show that unspecific seque nces within the 5' exon are particularly prone to inhibit both the for ward and the reverse first splicing step (branching). Using in vitro s election, we selected efficient 5' exons for the reverse branching rea ction. Precursor RNAs carrying these selected 5' exons reacted more ho mogeneously and faster than usual model precursor transcripts. This su ggests that unfavorable structures induced by the 5' exon can introduc e a folding step that limits the rate of in vitro self-splicing. These results stress how critical is the choice of the sequences retained o r 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 res pect to splicing than previously envisioned.