Examination of the folding pathway of the antigenomic hepatitis delta virus ribozyme reveals key interactions of the L3 loop

摘要

With the goal of gaining insight into the tertiary structure of the hepatitis delta virus ribozyme, cross-linking experiments using 4-thiouridine residues introduced in either the 5′-end portion of the substrate, or at seven strategic positions within the ribozyme, were performed. Mapping of the newly formed covalent bonds in cross-linked species obtained under various conditions, as well as using several mutated ribozymes, permitted monitoring of the formation of the ribozyme–substrate complex as the ribozyme proceeded along the folding pathway. In order to aid visualization of the tertiary structure transformation, an in silico animation of the “on” folding pathway was developed. In combination with those of the cleavage assays of structured substrates, these data shed light on the key contribution of the L3 loop in the formation of an active tertiary complex.