Increased ribozyme activity in crowded solutions

Abstract

Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg(2+) concentrations. Here, we used an RNA cleavage assay to show that the PEG and Ficoll crowder molecules increased the biochemical activity of the ribozyme, whereas sucrose did not. Crowding lowered the Mg(2+) threshold at which activity was detected and increased total RNA cleavage at high Mg(2+) concentrations sufficient to fold the RNA in crowded or dilute solution. After correcting for solution viscosity, the observed reaction rate was proportional to the fraction of active ribozyme. We conclude that molecular crowders stabilize the native ribozyme and favor the active structure relative to compact inactive folding intermediates.

Keywords: Macromolecular Crowding; PEG; RNA Catalysis; RNA Folding; Ribozyme; X-ray Scattering.

FIGURE 1.

Scheme of RNA cleavage reaction of Azoarcus group I ribozyme. a, RNA substrate (9 nucleotides; red) base pairs with the ribozyme internal guide sequence (IGS). The 3′-OH of an exogenous GTP (blue) bound in the ribozyme active site is the nucleophile that attacks the scissile phosphodiester of the substrate. The cleaved RNA products are released into solution. b, proposed folding pathway leading to active RNA and cleaved product. As [Mg²⁺] is raised, the unfolded (U) RNA goes through a collapse transition to a compact native-like intermediate (I_C). Further conformational rearrangements produce folded (F) and catalytically active native (N) structures.

FIGURE 2.

a, fraction of oligomer cleaved over time. ³²P-Labeled 9-nucleotide substrate was added to the prefolded L-3 Azoarcus ribozyme in 8% PEG 1000 solutions with varying Mg²⁺ concentrations as indicated. The amplitude of the initial phase is assumed to be proportional to the fraction of the native ribozyme. Data at 1.1 mm MgCl₂ were fit to a simple exponential for the initial phase (dashed line) and to a stretched exponential for the initial phase (solid line). Systematic deviations from the data at short times indicate that a simple exponential does not fully describe the data. We saw similar systematic differences under other solution conditions. No such systematic differences were observed for the stretched exponential fits (all other MgCl₂ concentrations). b, same as a but with 16% PEG 1000. Error bars were calculated as the S.D. of values from three repeats of four experimental conditions. Because these errors were found to be similar, the mean S.D. was assumed to represent the measurement error for all experiments.

FIGURE 3.

PEG stabilizes active ribozyme at low Mg²⁺. a, initial phase amplitude (A in Equation 1) of the first turnover versus MgCl₂ concentration. Amplitudes were obtained from Equation 1 fitted to data in Fig. 2 for 8% and 16% PEG and similar data for other PEG concentrations (not shown). Error bars represent experimental error propagated from Fig. 2. The lines are fits to a three-state Hill equation as in Refs. and and show how the ribozyme folds to its active state at lower MgCl₂ concentrations in crowded solutions. b, observed rate constant for substrate cleavage versus the amplitude of initial phase, normalized with the reciprocal of reported viscosities of PEG solutions at 298.15 K. These are as follows: 0% PEG, 0.89 centipoise; 4% PEG, 1.13 centipoise; 8% PEG, 1.46 centipoise; and 16% PEG, 2.39 centipoise (as interpolated from the data in Refs. and 28). Representative burst rates are 1.8, 2.1, 2.7, and 1.5 min⁻¹ for 0%, 4%, 8%, and 16% PEG 1000, respectively, at 2 mm MgCl₂. We omitted the data from experiments in 0% PEG 1000 at 3 and 5 mm MgCl₂ and in 8% PEG 1000 at 5 mm MgCl₂ as the reactions were too rapid to accurately measure the initial rate. Error bars represent errors propagated through the fits.

FIGURE 4.

Comparison of RNA folding measured by SAXS with folding as monitored by ribozyme activity. The activity data shown are same as in Fig. 3 for the initial phase amplitude (closed red circles). For comparison, the fraction of substrate cleaved at 25 s is shown (open red circles). The blue circles are the fraction cleaved at 20 s from Behrouzi et al. (20) and show good consistency between the studies.

FIGURE 5.

Effect of PEG molecular weight. Shown is the initial phase amplitude versus MgCl₂ concentration for solutions with different molecular weight PEG molecules as crowders. Data were fit as described for Fig. 3.

FIGURE 6.

Comparison of Ficoll and sucrose. a, fraction of oligomer cleaved as a function of time for the prefolded L-3 Azoarcus ribozyme in 5% Ficoll or 5% sucrose solutions. The lines and error bars are as described for Fig. 2. b, initial phase amplitude versus MgCl₂ concentration for 5% Ficoll and sucrose. For comparison, data in 0% and 4% PEG 1000 are also shown. The lines are fits to a three-state Hill equation.