Metal ions and the thermodynamics and kinetics of tertiary RNA folding

Divalent cations play a fundamental role in the stability and folding of tertiary RNAs. We have applied multiple spectroscopic, chemical and enzymatic probes to examine the cooperativity and stability of tertiary RNAs. We present a framework to quantify the free energy for tertiary RNA folding using Mg²⁺ and urea titrations. We describe the compaction process along the Mg-induced thermodynamic folding pathway. The kinetic pathway of this and other large RNAs is complex and often fraught with multiple kinetic traps. Intermediates can exist on certain pathways and folding can be under kinetic control. However, we show that a large ribozyme can fold all the way to the biological active state in 0.1 second (orders of magnitude faster than previously observed) without falling into kinetic traps. We introduce the Mg²⁺ and urea "chevron" plots and conduct the first complete, quantitative analysis of tertiary RNA folding pathway. A folding scheme containing two kinetic intermediates accounts for all the free energy, number of bound Mg²⁺ ions, and surface burial of the equilibrium transition. The folding of this ribozyme is best described by a classical pathway populated by discrete intermediates. These results indicate that the conformational search in tertiary RNA folding can be very fast and occur along a smooth energy landscape.