Pistol ribozyme cleavage products and the importance of the 2′-oxygen nucleophile at the cleavage site. (A) Mass spectrometry of the cleavage products from the A. putredinis bimolecular pistol ribozyme. Peaks corresponding to the expected 5′-cleavage (5′ clv) and 3′-cleavage (3′ clv) products are annotated, and the observed (obs.) and calculated (calc.) masses for these peaks are presented. (B) Catalytic strategies that can be used to promote the internal phosphoester transfer mechanism for RNA cleavage. Catalytic strategies include: α, the arrangement of the 2′-oxygen, phosphorus and 5′-oxygen atoms for in-line nucleophilic attack; β, neutralization of the negative charge on the nonbridging phosphate oxygen; γ, deprotonation of the 2′-hydroxyl group; δ, neutralization of the developing charge on the 5′-oxygen atom. (C) Ribozyme activity with an all-RNA substrate (G) (see also Fig. 1B) and a substrate analog wherein a 2′-deoxyguanosine is substituted for the guanosine ribonucleotide at position 8 of the substrate RNA (dG). Bimolecular reactions were incubated and analyzed as described in the legend of Figure 1B.
