Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa

Abstract

ABSTRACT Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium Paenibacillus polymyxa. We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg2+ concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the P. polymyxa pistol ribozyme – an aspect that has not been previously analysed for this ribozyme class. We determined that the P. polymyxa pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from P. polymyxa. We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.