The widespread occurrence of HDV-like ribozymes and their biochemical properties

The hepatitis delta virus (HDV) ribozyme represents but one of a class of RNAs capable of catalyzing the site-specific scission of their own phosphate backbone. This ribozyme was initially identified in the satellite virus that is its namesake, and subsequently isolated from an in vitro selection for self-cleaving ribozymes from the human genome. The latter ribozyme mapped to the second intron of the human CPEB3 gene, and was found conserved across mammalian CPEB3 genes but not the same gene in other species.;Comparative genomics on the HDV and CPEB3 ribozyme revealed several invariant positions situated within a nested double pseudoknot that were essential for HDV-like ribozyme catalysis. Using this information, structure-based descriptors were designed for use in high-throughput, bioinformatic searches. These searches revealed numerous putative HDV-like ribozymes, many of which possessed confirmed in vitro activity. These ribozymes were found in a wide array of organisms across nearly all the kingdoms of life, suggesting that HDV-like ribozymes, and likely ribozymes in general, are quite common in modern genomes.;Following initial identification and confirmation of in vitro activity for the new HDV-like ribozymes, their position in their host genomes was analyzed. Several of the identified ribozymes were found at the 5' end of retrotransposable elements. Subsequent bioinformatic searches uncovered many more retrotransposon-associated self-cleaving moieties, which were proven to process the 5' end of the transposable element from surrounding genomic space.;The initial structure-based searches revealed several HDV-like ribozymes containing non-canonical features. One of these sequences possessed a shortened helical domain typically thought invariant in HDV ribozymes, implying that the HDV and CPEB3 folds used to design the early descriptors did not represent the minimal fold needed for HDV-like self-cleavage. Subsequent mutational studies identified a 48-nucleotide motif that adhered to HDV self-scission requirements. This sequence served as the model from which to construct new structure-based descriptors that could be used to isolate minimal HDV-like ribozymes. These searches uncovered new ribozymes, including a third HDV-like family in the Branchiostoma floridae genome and several sequences obtained from human sewage samples. Many of these ribozyme adhered to typical HDV catalysis patterns; they exhibited a broad region of pH-independence and demonstrated a positive correlation between rate of scission and Mg 2+ concentration. However, the shortest identified sequence, drz-Mtgn-1, possessed several interesting kinetic traits.;Kinetic analysis of drz-Mtgn-1 revealed two distinct metal binding modes, only of one of which supports fast catalysis. This led to a bell-shaped metal response that was independent of the divalent metal provided. Furthermore, the ribozyme had markedly decreased activity in Ca2+, and calcium was shown to inhibit cleavage activity in a non-competitive manner when magnesium was also present in the reaction media. Analysis suggests this is due to unfavorable interactions calcium forms with the nucleotide immediately upstream of the cleavage site, while the parabolic metal-dependence is due to direct perturbations to the ribozyme core that occur as a result of excess stabilizing co-factors as the divalent metal concentration increases.