Kinetic Model of Mg2+ Induced RNA Tertiary Folding from Stopped Flow Fluorescence Data

Abstract

Many of the biological processes that involve RNA rely on its ability to form complex tertiary structures. The process of how these molecules fold into these tertiary structures is not well understood, in part due to the unpredictability and complexity of the interactions among nucleotides that define the tertiary structures. To better understand RNA folding we are investigating the prokaryotic rRNA GTPase center, a 60 nucleotide RNA which folds in to a complex tertiary structure in the presence of Mg2+ ions. Its tertiary interactions include formation of base triples, a triloop, as well as long-range nucleobase stacking interactions. To monitor its folding process we have replaced key adenine residues with the fluorescent base analog 2-aminopurine in different positions of this RNA to probe distinct local environments. Stopped flow kinetics techniques were used to examine the coordination of these different elements in the context of global folding. Data from the stopped flow experiments were used to refine a theoretical model of the folding pathway of this RNA. Our model consists of four temporally resolved kinetic steps with approximate rate constants. This work was funded by the NIH R01-GM098102 to KBH. Labeled RNA molecules were contributed by Agilent.