RNA Biophysics in Living Cells using Shape Chemistry

Abstract

Current knowledge of RNA structure and function has primarily been derived from studies carried out in dilute solution. However, the normal environment for RNA in cells is a highly crowded one, in which the concentration of macromolecules reaches 300 g/L. Global effects of synthetic crowding agents, such as polyethylene glycol, indicate that crowding can have dramatic effects on RNA structure. Studies under physiological conditions and at the level of individual nucleotides, however, are required to fully understand the biological structure-function relationships involving RNA. In this study, we use selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of a purine riboswitch RNA aptamer domain in healthy growing Escherichia coli cells. Our results identify nucleotide-resolution effects of the cytoplasm on RNA structure and show that the cellular environment indeed alters the structural properties of the purine riboswitch relative to dilute solution. Specifically, significant decreases in SHAPE reactivity are seen in the purine binding pocket in cells compared to dilute solution. We conclude that in cells, the purine aptamer exists in a less flexible, more structured conformation. Such structural differences can have consequences on ligand binding properties, such as equilibrium dissociation constants. Ultimately, our results show that dilute solution experiments only partially recapitulate the cellular environment and it is critical to consider the effects of macromolecular crowding to accurately understand RNA structure and function in cells.