Conformational dynamics that regulate the folding of group I intron ribozymes

Abstract

Ribonucleic acids (RNA) are an important class of biopolymers that execute widely different biological functions ranging from regulation of gene expression to biosynthetic catalysis. We hypothesize that RNA molecules sharing a conserved topological and functional fold but differing in their primary sequence, size and overall structural complexity, share a common kinetic folding mechanism and that certain preferred folding pathways exist to avoid misfolding. Our model system to investigate this hypothesis comprises of group I intron ribozymes that catalyze their own excision from precursor RNA molecules. Crystal structures available for three phylogenetically diverse group I intron ribozymes reveal a highly super-imposable arrangement of their core active sites generated by a conserved pattern of tertiary contacts.;We use time resolved hydroxyl radical (·OH) footprinting to explore the kinetic folding pathways of the three group I ribozymes under identical solution conditions. The data from ·OH footprinting experiments are fit to kinetic equations to generate optimal 'structural-kinetic models' that define the dominant folding pathway, rates of interconversion of folding intermediates, their structures and life times in solution. We validate these models through ribozyme activity assays.;We find that the folding rates of the homologous ribozymes are not dictated by their size or overall structural complexities but rather by the strength of their constituent tertiary motifs which govern the structure, stability and lifetime of the folding intermediates. Strong canonical tertiary interactions formed early during the folding reaction result in long lived intermediates that interchange structural features, thereby slowing their rates of folding to the native conformation. Early formation of non-canonical weaker tertiary interactions generate less stabilized folding intermediates that fold faster to the native state along mutually independent pathways. Our studies reveal that the dominant folding flux, for each ribozyme, proceeds through at least one optimally structured kinetic intermediate which provides sufficient stability to act as a nucleating scaffold and yet, retains enough conformational freedom to avoid kinetic trapping.;To facilitate high throughput single nucleotide resolution RNA structure probing, we developed a fluorescent primer extension and capillary electrophoresis based method. The collaboratively developed software CAFA (Capillary Automated Footprinting Analysis) enables rapid quantitative data analysis.