Hairpin Ribozyme - Structure

Structure

The minimal hairpin ribozyme-substrate complex folds into a secondary structure that includes two domains, each consisting of two short base paired helices separated by an internal loop. Domain A (helix 1 - loop A - helix 2) contains the substrate and the primary substrate-recognition region of the ribozyme. Domain B (helix 3 - loop B - helix 4) is larger and contains the primary catalytic determinants of the ribozyme. The two domains are covalently joined via a phosphodiester linkage that connects helix 2 to helix 3. These domains must interact with one another in order for catalysis to occur.

When the minimal ribozyme-substrate complex is allowed to fold under conditions of low ionic strength, the two domains stack one atop the other, forming an inactive, extended structure that resembles a hairpin. In order for catalysis to occur, the two domains lie parallel to one another in a fold that resembles a paperclip. In various publications, this RNA has been termed either the "paperclip" or "hairpin" ribozyme. Despite the fact that the former name has proven to be more accurate, the latter has become the commonly accepted nomenclature. In the laboratory, a functional interaction between the two domains is promoted by the addition of cations, whose positive charge suffices to overcome the electrostatic repulsion of the negatively charged RNA backbone. In nature, association of the two domains is assisted through a combination of metal ions (including Mg2+) and the presence of two additional helical domains that are not present in the minimal ribozyme-substrate complex but serve to promote proper three-dimensional folding. These additional domains stack upon helices 2 and 3, thereby promoting the association of the two functional domains through what is termed a four-way helical junction.

The structure and activity of the hairpin ribozyme has been explored using a wide range of complementary experimental methods, including nucleotide replacement, functional group substitution, combinatorial selection, fluorescence spectroscopy, covalent crosslinking, NMR analysis and x-ray crystallography. These studies have been facilitated by the ability of the functional complex to self-assemble from segments made by solid phase chemical RNA synthesis, permitting the incorporation of a wide variety of modified nucleotides that are not naturally found in RNA. Together, the results of these experiments present a highly congruent picture of the catalytic cycle, i.e. how the hairpin ribozyme binds its substrate, folds into a specific three-dimensional structure, catalyzes the reaction, and releases the product(s) of the reaction.