Folding of Bacterial Group I Ribozyme in Crowded Solutions

Abstract

Crowder molecules in solution alter the equilibrium between folded and unfolded states of biological macromolecules. It is therefore critical to account for the influence of these other molecules when describing the folding of RNA inside the cell, which contains up to 30% by volume of macromolecules.In this contribution we report the results of small angle x-ray scattering experiments on a catalytic RNA, or ribozyme, from the bacterium Azoarcus sp BH72 in the presence of polyethylene-glycol 1000 (PEG-1000), a molecular crowder with average molecular weight 1000 Da. We observe that, in agreement with expected excluded volume effects, the presence of PEG shifts the equilibrium to favor more compact RNA structures. Two observations highlight the stabilization of the compact RNA: firstly, the transition from the unfolded to folded (more compact) state occurs at lower MgCl2 concentrations in PEG; secondly, the radius of gyration of the unfolded RNA decreases from 76 A to 64 A as the PEG concentration increases from 0 to 20 % wt./vol.We discuss several physical effects by which the crowder molecules can possibly influence the RNA folding. These include modification of ionic activity, modification of water activity, and the excluded volume effect. We present evidence that whilst all of the above effects probably change the RNA folding with respect to a dilute aqueous solution, the shift in Mg2+ dependence of folding transition cannot be explained entirely by the change in ion activity. We conclude that the dominant influence is likely to be the excluded volume effect.