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

Abstract

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 59-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.

Developing Three-Dimensional Models of Putative-Folding Intermediates of the HDV Ribozyme

“…After the pseudoknot I.I formation, the geometry of the coaxial helices of the HDV ribozyme was fixed and the 5′ extremity was docked inside the catalytic core in over 95% of the structures obtained ( Figure 3B). This position was in agreement with the data from a previous crosslinking analysis (Reymond et al, 2007). In the remaining structures, the 5′ region was generally positioned outside of the catalytic core, and, in a few exceptional cases, positioned through the formation of a kink turn located at the scissile phosphate bond.…”

“…Specifically, the tertiary constraints sufficient to define an intermediate in the MC-Sym software are those that correspond to the interactions that are disrupted in the mutants previously used in the analysis of the folding pathway (Reymond et al, 2009a) ( Figure 2B). In addition, the distances between two nucleotides shown to be in close proximity by UV induced covalent bonds in previous crosslinking studies were found to be present in these predicted structures (Ouellet et al, 2004;Reymond et al, 2007). After minimization, all of the putative structures obtained for a given intermediate were visualized using the Visual Molecular Dynamic software (VMD) (Humphrey et al, 1996), and the most representative structure was selected based on both its stability and the average root mean square deviation ( Figure 2C).…”

A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme

“…All of the ribozymes and the RzA strands were synthesized by run-off transcription as described previously ( 19 ). Briefly, RNA molecules were produced by annealing two strands of complementary DNA oligonucleotides that included the T7 RNA promoter followed by the sequence of the desired ribozyme.…”

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

“…This important characteristic has been fully exploited in order to decipher the folding pathway of the HDV ribozyme, by far the most complex folding pathway elucidated to date for a small ribozyme. Extensive work has been performed using a number of approaches, including in vitro selection and photo-induced cross linking, in order to identify a specific set of mutants able to halt the folding pathway at each stable intermediate [ 39 , 49 ]. Briefly, after the recognition of the substrate by the ribozyme leading to the formation of the stem I, five subsequent conformational steps are required in order to form the catalytically active structure (Fig.…”

“…At the beginning of the folding pathway, the loop III and junction I/IV are located relatively far from each other and have a significant amount of freedom. The formation of the pseudoknot I.I is required in order to bring together these two single-stranded regions, thereby trapping the substrate within the catalytic core [ 49 ]. This requires an important entropic loss, and the driving force of this step is most likely the formation of two GC base pairs and the coaxial stacking of the helices I–I.I–IV.…”