Sequence, structure, and activity of pistol ribozymes. (A) Consensus sequence and secondary structure model for pistol ribozymes based on the alignment of 500 unique representatives found in DNA sequence databases comprised of complete bacterial genomes, microbial metagenomic DNA, and bacteriophage DNA. The arrowhead designates the ribozyme-mediated cleavage site. (B) (Top) Sequence and predicted secondary structure of the bimolecular pistol ribozyme construct derived from A. putredinis. Nucleotides in red correspond to the highly conserved positions from the consensus model. (Bottom) Self-cleavage activity of the A. putredinis bimolecular ribozyme construct. The 32P-labeled substrate RNA (∼1 nM) was incubated for the times indicated at 23°C in the presence (+) or absence (−) of 20 mM MgCl2 and unlabeled enzyme RNA (∼100 nM) (see Materials and Methods for details). The cleavage products were separated by denaturing 20% PAGE. The full-length substrate (sub) and 5′-cleavage product (5′ clv) are denoted. (C) Sequence, secondary structure, and activity of a bimolecular pistol ribozyme construct from P. polymyxa. Annotations are as described in A and B. (D) Sequence, secondary structure, and activity of a bimolecular pistol ribozyme construct from E. bacterium. Annotations are as described in A and B.
