Biochemical analysis of pistol self-cleaving ribozymes

Abstract

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min −1 under physiological Mg 2+ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2 ′ -oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.

Crystal structure of Pistol, a class of self-cleaving ribozyme

“…Our structure reveals the nucleobases that are likely to be involved in the internal transesterification reaction of Pistol. The structure validates prior biochemical results of the Pistol self-cleavage mechanism and further elucidates additional mechanistic details that cannot be easily addressed with biochemical analysis (10). The structure shows that Pistol adopts an overall compact fold stabilized by the A-minor motif commonly found in many RNA structures, which explains the high sequence conservation for the stretch of adenines found in Pistol.…”

“…Currently, the classes of self-cleaving ribozymes consist of Hammerhead, Hairpin, Hepatitis Delta Virus ribozyme (HDV), Varkud Satellite (VS), glmS ribozyme, Twister, Twister sister, Hatchet, and Pistol (10). These classes differ based on their size, structure, and cleavage mechanism.…”

“…Biochemical analysis reveals that Pistol can use a variety of divalent metal ions to carry out a complete site-specific, self-cleaving reaction, whereas utilization of monovalent cations results in modest cleavage rates (3,10). The rate of Pistol self-cleavage has been estimated to be >10 min −1 under physiological conditions and >100 min −1 under optimal magnesium and pH conditions (10). Pistol self-cleavage is via an internal transesterification reaction, in which the substrate RNA of Pistol G10 2′-hydroxyl on the ribose makes a nucleophillic attack on the adjacent 3′-phosphate (Fig.…”

“…1B) (3, 10). Self-cleaving ribozymes can enhance the rates of the internal transesterification reaction by using catalytic strategies, such as deprotonation of the 2′-hydroxyl group and neutralizing the negative charge on the nonbridging oxygen of the scissile phosphate or 5′-oxygen of the cleaved substrate (10,(13)(14)(15).…”

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes

“…I shall also tentatively place the new pistol ribozyme (13,60) into the same group. The pH dependence of this ribozyme is similar to that of the hammerhead.…”