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

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.

Keywords: Paenibacillus polymyxa ATCC 842; RNA World; RNA catalysis; apparent first-order rate constant; general acid-base catalysis; internal phosphoester transfer; nucleolytic ribozymes; pKa of hydrated metal ion; self-cleaving ribozymes; transesterification.

Graphical abstract

Figure 1.

Sequence, structure and activity of the P. polymyxa pistol ribozyme. (A) Sequence and secondary structure of the P. polymyxa pistol ribozyme. Nucleotides in red correspond to the highly conserved positions from the consensus model [14]. Loop 3 (nucleotides coloured grey) is absent in the bimolecular construct (see Supplementary Fig. 1A for details). Altered nucleotides in the mutant construct are boxed. P1-, P2- and P3-stems are indicated. Cleavage site is indicated by a black arrowhead. (B) Self-cleavage activity of the P. polymyxa pistol ribozyme. Internally labelled cleavage products were separated by denaturing 20% PAGE. The full-length ribozyme (FL) and cleavage products (5ʹ clv; 3ʹ clv) are indicated by black arrowheads. T1: RNase T1-ladder; OH⁻: partial alkaline digest-ladder (see Materials and methods for details). WT: wild-type ribozyme; MT: mutant ribozyme. (C) Cleavage activity of the P. polymyxa bimolecular pistol ribozyme construct. The ³²P-labelled substrate RNA (5 nM) was incubated for the times indicated in the presence (+) or absence (-) of 20 mM MgCl₂ and unlabelled enzyme RNA (100 nM) (see Materials and methods for details). Cleavage products were separated by denaturing 20% PAGE. The full-length substrate (sub) and 5ʹ-cleavage product (5ʹ clv) are denoted

Figure 2.

Metal ion dependency of the P. polymyxa pistol ribozyme cleavage. (A) Pistol ribozyme cleavage assays with the P. polymyxa bimolecular construct incubated for 30 min in the absence (-) or presence of various 1 mM divalent metal ions. (B) Dependence of the P. polymyxa pistol ribozyme cleavage rate on the pK_a of the hydrated divalent metal ion. Six divalent metal ions at the concentration of 1 mM, pH 6.0 and 2 M NaCl [reaction conditions according to 18] were tested. pK_a values are taken from . (C) Reactions of the P. polymyxa bimolecular construct in the absence (-) or presence (+) of 5 mM cobalt hexammine chloride [Co(NH₃)₆Cl₃] or MgCl₂ for 60 min in the presence of 5 mM EDTA. (D) Pistol ribozyme cleavage assays with the P. polymyxa bimolecular construct incubated for 60 min in the absence (-) or presence of various 1 M monovalent metal ions. To chelate contaminating divalent metal ions, 30 mM EDTA was added to these reactions. For the positive control (PC), 25 mM MgCl₂ was added

Figure 3.

Kinetic characteristics of the P. polymyxa pistol ribozyme cleavage. (A) Effect of Mg²⁺ on the rate of P. polymyxa pistol ribozyme cleavage. The log-log plot of k_obs values at pH 6.0 versus MgCl₂ concentrations ranging from 0.1 mM to 25 mM is shown. (B) Effect of pH on the rate of P. polymyxa pistol ribozyme cleavage. The log-log plot of k_obs values at the MgCl₂ concentration of 1 mM versus pH values ranging from 5.25 to 8.75 is depicted

Figure 4.

Ligation activity of the P. polymyxa pistol ribozyme. (A) Kinetic assay using the P. polymyxa bimolecular ribozyme construct for cleavage (see Materials and methods for details) at the MgCl₂ concentration of 5 mM and 30 mM Tris (pH 7.5). The corresponding time points are indicated. The cleavage products were separated by denaturing 20% PAGE. The full-length substrate (sub) and 5ʹ-cleavage product (5ʹ clv) are denoted. (B) Kinetic ligation assay of the P. polymyxa pistol ribozyme at the MgCl₂ concentration of 5 mM and 30 mM HEPES (pH 7.5). 15% PEG8000 was used as an additive to improve the assay quantification. The corresponding time points are indicated. The ligation products were separated by denaturing 10% PAGE. The full-length ribozyme (FL) and 5ʹ-cleavage product (5ʹ clv) are denoted. As a positive control, hot transcription of the wild-type (WT) and mutant (MT) P. polymyxa pistol ribozyme was performed. (C) Comparison of the cleavage and ligation activities of the P. polymyxa pistol ribozyme. Approach to equilibrium at 5 mM Mg²⁺ and pH 7.5 is shown. The dashed line represents the mean value of the fraction of the full-length ribozyme (ligation) or uncleaved substrate (cleavage) at equilibrium, f_eq = 0.07. Open grey circles indicate cleavage of the ligated ribozyme, which was detected during the ligation assay. The inset shows a zoom-in of the region between 0 min and 5 min of the ligation reaction