Interactions within and between the mRNA substrate and the ribozyme, and their treatment during computation. (A) Schematic of the substrate binding step in trans-splicing. The mRNA substrate is illustrated as a hairpin structure, and the body of the ribozyme is illustrated as a gray oval. Unfolding of the target site on the mRNA (first step) and release of the internal guide sequence (IGS) (second step) allow the mRNA target site and the IGS to hybridize (third step). (B) The 3D structure of the Tetrahymena ribozyme (Lehnert et al. 1996) indicates that the AAA linker (black) is not stacked on the P1 duplex. Additionally, the 3′-exon (gray) is close enough to the IGS (black) for interactions if the mRNA (gray) is absent. (C) To facilitate computational treatment of the binding process, the body of the ribozyme was replaced by a linker sequence joining the IGS to the 3′-exon. This linker does not include the natural AAA linker but consists of five unspecified nucleotides (N), which are ignored in the calculation of base-pairing and π-stacking interactions.
