title: Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme.
abstract: The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Tinsley
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D024202:Electrophoretic Mobility Shift Assay; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D007477:Ions / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013725:Terbium / Q000737:chemistry / Q000378:metabolism
chemical_list: D007477:Ions; D016337:RNA, Catalytic; D013725:Terbium; D008274:Magnesium