Effect of substrate recognition sequences on product formation.

(A) Kinetics of spliceozyme splicing in vitro, measured as the molar percentage of the initital substrate concentration (CAT pre-mRNA) converted to product (CAT mRNA). The reactions were analyzed as in Figure 2. Data from spliceozymes with a 3′-terminal P9.2 helix of 9 base pairs are shown with filled symbols, including spliceozyme 5′-termini of a 5′-duplex (triangles), P1 extension helix (circles), and P1 helix (squares). Empty symbols denote results from spliceozymes terminating in a P1 helix at the 5′-terminus, and the 3′-terminal P9.2 helix truncated to 8 base pairs (diamonds), 7 base pairs (squares), 6 base pairs (triangles), and 5 base pairs (circles). Error bars correspond to standard deviations from three experiments. (B) Sequences of splicing junctions resulting from RT-PCR, cloning and sequencing of trans-splicing products from the reaction with a P9.2 helix of 5 base pairs and a P1 helix at the 5′-terminus. Note that the wild type sequence (WT) differs from the splicing products in three silent mutations (empty triangles), which confirmed that the sequences were splicing products and not a contamination by the wild type gene. Nine out of ten sequences showed the sequence expected from correct splicing at the 3′-terminal G, whereas one sequence indicated that the guanosine four nucleotides downstream of the intended 3′-splice site was used instead. The sequence participating in the P1 helix is underlined, containing the 5′-splice site (filled triangle).