Kinetics and Mechanism of RNA Folding studied by SAXS

Abstract

RNAs fold into unique native structures to perform specific biological functions. Detailed identification of the kinetics of RNA-folding should provide insight to the nucleation and collapse mechanisms. The Azoarcus ribozyme exhibits two thermodynamic transitions: 1) U to Ic at low Mg2+ concentrations (U: unfolded state, Ic: compact intermediate state), ascribed to the assembly of double helices in the center of the RNA and 2) Ic to N at high Mg2+ concentration (N: native state), which involves further secondary-structural rearrangements to form the final folded structure including tertiary interactions. We have studied the rate of the folding transitions and the role of cooperative tertiary interactions in stabilizing the Ic and N states in order to understand the folding mechanism. The real-time dependence of folding of the wild type and mutant Azoarcus ribozyme as a function of cation concentration (Mg2+ or Ba2+) was monitored using time-resolved small angle X-ray scattering (SAXS) coupled with a stopped-flow sample system. At high Mg2+ concentrations the Azoarcus ribozyme collapses through multiple pathways, with a majority (∼ 90 %) following a fast folding route within t ∼ 10 ms and the rest collapsing at t > 3 min. The folding rate at short time scales decreases with decreasing cation concentration. The rate changes significantly in the range of the first transition, suggesting that initial cation charge screening of the repulsion of the negatively charged RNA chains plays an important role in determining the formation of tertiary interactions. Interestingly, Ba2+ and single-site mutations in the Azoarcus ribozyme lead to faster collapse of the RNAs on short timescales, suggesting that folding of the wild type in Mg2+ requires more time to stabilize the correct folded tertiary structure through the rearrangement of secondary structures.