Dynamic assembly of a large multidomain ribozyme visualized by cryo-electron microscopy.

Many RNAs rely on their 3D structures for function. While acquiring functional 3D structures, certain RNAs form misfolded, non-functional states ('kinetic traps'). Instead, other RNAs sequentially assemble into their functional conformations over pre-folded scaffolds. Elucidating the principles of RNA sequential assembly is thus important to understand how RNAs avoid the formation of misfolded, non-functional states. Integrating single-particle electron cryomicroscopy (cryo-EM), image processing, in solution small-angle X-ray scattering (SAXS), EM-driven molecular dynamics (MD) simulations, structure-based mutagenesis, and enzymatic assays, we have visualized the sequential multidomain assembly of a self-splicing ribozyme of biomedical and bioengineering significance. Our work reveals a distinct dynamic interplay of helical subdomains in the ribozyme's 5'-terminal scaffold, which acts as a gate to control the docking of 3'-terminal domains. We identify specific conserved and functionally important secondary structure motifs as the key players for orchestrating the energetically inexpensive conformational changes that lead to the productive formation of the catalytic pocket. Our work provides a near-atomic resolution molecular movie of a large multidomain RNA assembling into its functionally active conformation and establishes a basis for understanding how RNA avoids the formation of non-functional 'kinetic traps'.