RNA Folding in Crowded Solutions

Abstract

Many non-coding RNAs fold into specific three-dimensional structures in order to act as catalysts or regulatory elements. Folded RNA structures are stabilized by K+ and Mg2+ ions that neutralize the phosphate negative charge and bind to specific RNA motifs. In the cell, however, the stability of the folded RNA depends on its interactions with many other solutes. Using small angle X-ray scattering to measure the folding of a bacterial group I ribozyme, we showed that molecular crowding equivalent to what is present in real cells stabilizes RNA tertiary structures by several kcal/mol. We used polyethylene glycol (PEG) with different molecular weights as a crowding agent, as it does not appreciably interact with the RNA. Stabilization of the native RNA in MgCl2 or NaCl is most likely due to the excluded volume effect, and as our results were not explained by changes to water or ion activity. Crowder molecules favor more compact structures of the unfolded and native RNAs. Furthermore, compression of the native state ensemble correlates with an increase in ribozyme catalytic activity. Consequently, the ribozyme reaches its catalytically active structure at much lower Mg2+ concentrations in a crowded milieu than in a dilute solution. Thus, our results partly explain why many ribozymes are more active in the crowded environment of the cell than in the test tube.