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

Many RNAs rely on their 3D structures for function. To acquire functional 3D structures, certain RNAs form misfolded states (‘kinetic traps’), while other RNAs sequentially assemble onto well-folded scaffolds. Elucidating the principles of RNA sequential assembly is thus important to understand how RNAs avoid forming non-functional states. Integrating single-particle cryo-EM and image processing, SAXS, and molecular simulations, we have visualized the sequential multidomain assembly of a self-splicing ribozyme of biomedical and bioengineering significance. Our work reveals 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 residues and secondary structure motifs as the key players to sequentially orchestrate these structural transitions and ensure the formation of the catalytic pocket. Our work provides an unprecedented 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’.