Conformational dynamics in folding and function of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is a unique structural motif required for replication of a human pathogen Hepatitis Delta virus. To accomplish the cleavage rate enhancement, the ribozyme utilizes multiple catalytic strategies. In the present work, a combination of experimental and theoretical techniques was applied to study the reaction mechanism and to characterize conformational changes on global, local, nucleotide, and atomic levels. Using fluorescence resonance energy transfer (FRET), we examined the FRET ratios of our precursor complexes varying in 5' sequence. Our observations highlight the large influence that a single or few nucleotides of 5' substrate sequence have on global structure, as well as on the catalytic activity, of the trans-acting HDV ribozyme.; The terbium(III) ion was used to footprint the precursor form of the cis-acting HDV ribozymes varying in 5' sequence. Our experiments revealed that the conformation of the catalytic pocket depends on the identity of the -1 nucleotide. The compact structure of the catalytic pocket in the ground state resembling the product structure results in a most active ribozyme. Our molecular dynamics simulations on both precursor and product forms of the ribozyme complement the existing structural and biochemical data are most consistent with the conformational changes invoked for the general base catalysis mechanism. The active site of the precursor accommodates a magnesium ion interacting with reactive groups through a complex network of water molecules.; In one simulation our trajectory analysis revealed a rapid rotation of G76 towards the catalytic pocket, followed by the formation and long-term retention of hydrogen bonds with stem P1. To experimentally test for the plausibility of such interactions, we performed second-site mutagenesis experiments. The deleterious G76A mutation in a trans-acting HDV ribozyme is compensated for by mutations in P1 that complement the hydrogen bonding pattern of adenosine, supporting a functional role for an interaction between 76 position and P1 in this form of the HDV ribozyme.; In summary, the presented biochemical and biophysical studies provide new insights into the relationship between function and conformational dynamics in the reaction catalyzed by the HDV ribozyme.