Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (Rg) decreases from 75 Å to 55 Å. A further decrease in Rg to 45 Å occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55–45 Å compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose Rg is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establish the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.