Structure and activity assays with hatchet construct Ht-2. (A) Sequence and secondary structure for the bimolecular construct Ht-2 derived from a metagenomic sequence. Mutations (M1, M3, and M5) and compensatory mutations (M2 and M4) that restore structure are boxed and occur at the locations indicated. Other annotations are as described in the legend to Figure 1B. (B) Ribozyme activities of WT or mutant Ht-2 constructs using a 5′ 32P-labeled substrate RNA. (NR) The reaction with WT RNA at time 0 as prepared by adding stop buffer before the addition of Mg2+. The asterisk denotes a reaction mixture without added enzyme strand. Other annotations and reaction conditions are as described in the legend to Figure 1C. (C) An example of a time course assay for ribozyme cleavage used to establish a kobs value under a single reaction condition. (D) Plot representing the dependence of kobs on Mg2+ concentration. (E) Plot representing the dependence of kobs on pH. Three different buffers were used to span the pH range from 5 to 9.
