Molecular recognition in a trans excision-splicing ribozyme: Non-Watson-Crick base pairs at the 5′ splice site and ωG at the 3′ splice site can play a role in determining the binding register of reaction substrates

Trans excision-splicing (TES) ribozymes, derived from a Pneumocystis carinii group I intron, can catalyze the excision of targeted sequences from within RNAs. In this report, the sequence requirements of the splice sites are analyzed. These conserved sequences include a u-G wobble pair at the 5′ splice site and a guanosine in the ω position at the 3′ splice site (in the substrate). We report that 7 out of 16 base pair combinations at the 5′ splice site produce appreciable TES product. This promiscuity is in contrast to results reported for analogous self-splicing reactions using a Tetrahymena ribozyme. At long reaction times TES products dissociate and rebind free ribozyme, at which point product degradation occurs via the 5′ cleavage reaction. Unexpectedly, only in cases where Watson-Crick base pairs form at the 5′ splice site do we see degradation of TES products at cryptic sites, suggesting that non-Watson-Crick base pairs at the 5′ splice site are acting in concert with other factors to precisely determine the binding register of TES reaction substrates within the ribozyme. Moreover, cryptic site degradation does not occur with the corresponding reaction substrates, which additionally contain ωG, suggesting that ωG can play a similar role. We report that ωG cannot be replaced by any other base, so TES substrates require a guanosine as the last (or only) base to be excised. Additionally, we demonstrate that P9.0 and P10 are expendable for TES reactions, suggesting that ωG is sufficient as a 3′ molecular recognition element.