Efficient ligation of the Schistosoma hammerhead ribozyme

Abstract

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.

Figure 1

Sequence and secondary structure of the trans Schistosoma hammerhead ribozyme constructs. The full construct is shown for Schist26, whereas only stem III and the corresponding P1 are shown for Schist24 through Schist20 since the rest of the construct is identical to Schist26. Construct names correspond to the number of bases in the substrate strand. The double-headed arrow indicates the tertiary loop-loop interaction between stems I and II. Canonical base pairs are shown as solid lines and the Hoogsteen base pair is shown with a ●. The conserved nucleotides that make up the catalytic core are shown in outline and numbered according to standard convention. Bold nucleotides indicate the ribozyme strand and the single headed arrow points to the scissile bond. The cleavage experiments employ a two-stranded construct consisting of the ribozyme and the full-length substrate. The ligation experiments employ a three-stranded construct consisting of the ribozyme, the 5’-cleavage product, P1, which has a 2', 3' cyclic phosphate at C¹⁷, and the 3’-cleavage product, P2, which has a 5’-OH on C¹⁸. For the UUCG control, the sequence in the stem II loop is replaced with a UUCG sequence (boxed).

Figure 2

Reaction scheme of the trans hammerhead ribozyme cleavage and ligation reaction. R is the ribozyme strand, S is the substrate strand, and P1 and P2 are the 5' and 3' cleavage products, respectively. k_cleave is the cleavage rate constant, k_ligate is the ligation rate constant, k_off is the P1 dissociation rate constant, k_on is the P1 association rate constant. K_eq^int is the equilibrium constant for the cleavage-ligation step of the reaction, and K_diss is the equilibrium constant for dissociation of P1. The asterisks indicate the catalytically active forms of the cleaved and ligated complexes.

Figure 3

Single turnover ligation kinetics of the Schist26 hammerhead ribozyme. A) A 20% denaturing polyacrylamide gel used to separate ³²P-labeled ligated full-length substrate from ³²P-P1 in the ligation experiment for Schist26 in 10 mM Mg²⁺. The different lanes correspond to samples removed from the ligation reaction at various times after addition of Mg²⁺. B) Plot of the fraction of ligated substrate vs. time where the solid line shows the fit to a single exponential with a k_{obs, ligate} of ~26 min⁻¹.

Figure 4

Fraction of cleaved substrate in cleavage and ligation experiments. Plots of the fraction of cleaved substrate vs. time from the cleavage (circles) and ligation (squares) experiments in A) 1 mM Mg²⁺ and B) 5 mM Mg²⁺. C) Maximum fraction of cleaved substrate observed in the cleavage experiments used to determine k_{obs, cleave} (circles) and in the ligation experiments used to determine k_{obs, ligate} (squares) at varying Mg²⁺ concentrations.

Figure 5

Mg²⁺ dependence of the kinetics for the Schist26 and Schist21 constructs. A plot of k_{obs, cleave} for Schist26 (circles), k_cleave for Schist21 (squares), and calculated k_ligate (diamonds) vs. Mg²⁺ concentration. For k_{obs, cleave} and k_cleave the lines are fits of the Mg²⁺ dependence of the rate constant, k, to a two-state model for Mg²⁺ binding using the equation k = k_max [Mg²⁺]/([Mg²⁺] + [Mg²⁺]_1/2), where k_max is the rate constant at saturating Mg²⁺, and [Mg²⁺]_1/2 is the Mg²⁺ concentration required to achieve half the maximal cleavage rate constant. This analysis assumes a Hill coefficient of 1 since there was no evidence for cooperative binding of Mg²⁺ for any of the kinetic data. These fits yield [Mg²⁺]_1/2 of 55 ± 10 mM for k_{obs, cleave} for Schist26 and 17 ± 3 mM for k_cleave for Schist21. The k_ligate does not saturate at the Mg²⁺ concentrations employed here making it impossible to determine a [Mg²⁺]_1/2. Thus for k_ligate the solid line is a simple linear fit of k_ligate with [Mg²⁺]. The error bars are based on a 20% error between replicate experiments for the measured rate constants (see Table 1).

Figure 6

Native polyacrylamide gels of ³²P-labeled P1 for pulse-chase experiments used to measure rate constants for P1 dissociation from the ribozyme complex in 1 mM Mg²⁺. The times indicate when the sample was loaded on the gel after the unlabeled chase was added to the reaction. The samples were loaded on a running gel, so the bands are shifted up in the gel for the longer time points. The left half of the gel is data for the P1 of Schist23 dissociating from the Schist23 ribozyme and the right half of the gel is for the P1 of Schist24 dissociating from the Schist26 ribozyme (see Methods).

Figure 7

Single turnover cleavage kinetics followed for 24 hrs to monitor conversion of inactive to active species for Schist26. A plot of the fraction of cleaved substrate vs. time is shown for cleavage experiments in 0.1 mM (circles), 1 mM (squares), and 10 mM (open diamonds) Mg²⁺. The plots are a combination of the cleavage data from the 24-hr experiments and from the manual and quench flow experiments. The data are fit to a double exponential, where one exponential corresponds to the cleavage/ligation kinetics and the second exponential corresponds to kinetics for the conversion of inactive to active ribozymes. The fit to the second exponential yields k_{inactive→active} of ~0.006 min⁻¹.

Figure 8

Single turnover cleavage kinetics for the cave cricket hammerheads. A plot of the fraction of cleaved substrate vs. time for Dolichopoda baccettii (solid symbols) and D. schiavazzii (open symbols) cricket hammerhead constructs at 0.5 mM (squares), 1 mM (circles) and 5 mM (triangles) Mg²⁺.