Structure and cleavage activity of AspLC. Secondary structure diagrams of the active forms of (A) Allovahlkampfia AspLC-169.28 and (B) Naegleria pringsheimi NprLC-191.28. Substitutions in AspLC compared with NprLC are indicated in red. Helical indels are boxed. (C) Time-course cleavage analysis of AspLC-169.28 and NprLC-191.28 transcripts separated on 5% urea polyacrylamide gels. Pre, LC precursor RNA; 5′LC, 5′ cleavage product containing the LC ribozyme sequence. (D) Kinetic cleavage analysis of AspLC-169.28 and NprLC-191.28 transcripts presented as fraction of uncleaved precursor versus time. (E) Primer extension analysis of the same sample of AspLC-169.28 analyzed in C and D, but tracking the 3′ cleavage product. BP, branch point nucleotide; IPS, internal processing site. A sequencing ladder made by sequencing of the AspLC-169.28 plasmid construct with the same primer (C720) as used for primer extension is shown. Interpretations of IPS and branch point are presented below the primer extension analysis. (F) Schematic presentation of the lariat capped 3′ cleavage product (I-Asp I mRNA) and released 5′ cleavage product (AspLC ribozyme sequence).